[go: up one dir, main page]

WO2011111562A1 - Procédé de création d'un agrégat cellulaire tridimensionnel, d'un support de gel tridimensionnel pour la culture cellulaire utilisé dans ce procédé, et agrégat cellulaire tridimensionnel - Google Patents

Procédé de création d'un agrégat cellulaire tridimensionnel, d'un support de gel tridimensionnel pour la culture cellulaire utilisé dans ce procédé, et agrégat cellulaire tridimensionnel Download PDF

Info

Publication number
WO2011111562A1
WO2011111562A1 PCT/JP2011/054573 JP2011054573W WO2011111562A1 WO 2011111562 A1 WO2011111562 A1 WO 2011111562A1 JP 2011054573 W JP2011054573 W JP 2011054573W WO 2011111562 A1 WO2011111562 A1 WO 2011111562A1
Authority
WO
WIPO (PCT)
Prior art keywords
dimensional
gel
temperature
polymer gel
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2011/054573
Other languages
English (en)
Japanese (ja)
Inventor
満 明石
隆彬 麻生
泰治 荘村
卓也 松本
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Osaka NUC
Original Assignee
Osaka University NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Osaka University NUC filed Critical Osaka University NUC
Publication of WO2011111562A1 publication Critical patent/WO2011111562A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/087—Acrylic polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • 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
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—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

Definitions

  • the present invention relates to a method for producing a three-dimensional cell aggregate, a three-dimensional gel carrier for cell culture used therefor, and a three-dimensional cell aggregate.
  • Patent Document 1 a method using a cell sheet for the induction of tissue regeneration without using a scaffold has been developed.
  • cells are cultured using a support material whose surface is coated with a polymer or copolymer whose upper critical solution temperature or lower critical solution temperature is in the range of 0 to 80 ° C., and then the upper critical solution temperature.
  • the cell sheet is recovered by peeling from the support material at a temperature equal to or higher than the lower critical solution temperature.
  • the present invention solves the above problems and provides a method for producing a three-dimensional cell aggregate that can provide a three-dimensional cell aggregate that can be used as a tissue regeneration transplant material having any shape without complicated operations.
  • Another object of the present invention is to provide a three-dimensional gel carrier for cell culture used therefor and a three-dimensional cell aggregate of an arbitrary shape obtained by the production method thereof.
  • the present inventors have intensively studied. As a result, cells are seeded and cultured on a molded body of a hydrophilic polymer gel capable of changing volume, and the volume of the hydrophilic polymer gel is changed.
  • the present invention has been completed by finding that cultured cells can be detached as a three-dimensional cell aggregate. That is, the method for producing a three-dimensional cell assembly of the present invention uses a three-dimensional molded body of a hydrophilic polymer gel capable of changing volume as a carrier, seeds and cultures the cells on the carrier, and changes the volume of the carrier. In this case, the cultured cells to which the three-dimensional structure is imparted are detached as a three-dimensional cell aggregate.
  • thermophilic polymer gel it is preferable to use a temperature-responsive polymer gel as the hydrophilic polymer gel.
  • the temperature-responsive polymer gel is a hydrophilic polymer gel having a lower critical solution temperature (LCST) that shrinks at a critical temperature (Tc) or higher, and the lower critical solution temperature. It is preferable that the cells are cultured at a higher temperature, and the cultured cells are detached from the hydrophilic polymer gel at a temperature equal to or lower than the lower critical solution temperature.
  • the temperature-responsive polymer gel is preferably a homopolymer or copolymer cross-linked product of N-alkyl-substituted (meth) acrylamide.
  • the hydrophilic polymer gel using a resin matrix produced by using a three-dimensional printing method.
  • another invention of the present invention provides a three-dimensional gel carrier for cell culture used in the above-mentioned method for producing a three-dimensional cell aggregate, and comprises a three-dimensional molded body of a hydrophilic polymer gel capable of changing volume. It is characterized by being used as a template for imparting a three-dimensional structure to cultured cells.
  • the hydrophilic polymer gel is preferably a temperature responsive polymer gel.
  • the temperature-responsive polymer gel is preferably a homopolymer or copolymer cross-linked product of N-alkyl-substituted (meth) acrylamide.
  • another invention of the present invention provides a three-dimensional cell aggregate produced by using the above-described method for producing a three-dimensional cell aggregate, and a three-dimensional molded article of a hydrophilic polymer gel capable of changing volume. It is characterized by comprising an aggregate of cells that are cultured with a template and given a three-dimensional structure by the template.
  • the cultured cells can be detached as a three-dimensional cell aggregate by expanding the hydrophilic polymer gel, so that the three-dimensional cell aggregate can be produced without complicated operations. It becomes possible. Further, since the hydrophilic polymer gel can be formed into an arbitrary shape, a three-dimensional cell aggregate having an arbitrary shape can be produced by using the hydrophilic polymer gel as a carrier. In addition, a three-dimensional cell aggregate capable of reproducing a living tissue can be produced.
  • A is a gel using polyethylene glycol dimethacrylate (PEG-DMA) as a crosslinking agent
  • B is a crosslinking agent using N, N′-bisacrylamide (MBAA).
  • 10 is a photograph showing chondrocyte differentiation in Experimental Example 3. It is a photograph which shows the calcification in Experimental example 3.
  • 6 is a photograph showing the expression of a hypoxic system (HIF-1 ⁇ ) in Experimental Example 4.
  • 6 is a photograph showing the expression of VEGF in Experimental Example 4.
  • 6 is a photograph showing formation of new bone in Experimental Example 5.
  • the method for producing a three-dimensional cell aggregate of the present invention uses a molded article of a hydrophilic polymer gel having a three-dimensional structure and a changeable volume as a carrier, seeded and cultured on the carrier, and the volume of the carrier.
  • the cultured cells to which the three-dimensional structure is imparted are peeled off as a three-dimensional cell aggregate by changing the above.
  • three-dimensional cell aggregate refers to an aggregate of cells having a thickness of a single layer or more and having an arbitrary three-dimensional shape.
  • Arbitrary three-dimensional shapes refer to all shapes including planar shapes such as films and sheets, rectangular shapes, spherical shapes, columnar shapes, curved shapes, and the like.
  • the “cell sheet” used in the present specification is an aggregate of single-layered cells, and refers to one having only a two-dimensional spread.
  • the hydrophilic polymer gel used in the present invention is obtained by crosslinking a hydrophilic polymer with a crosslinking agent, and has a three-dimensional structure and can be changed in volume.
  • the hydrophilic polymer gel used in the present invention serves not only as a carrier for culturing cells but also as a template for imparting a three-dimensional structure to cultured cells.
  • “volume changeable” means that in an aqueous solution, the volume changes in response to an external stimulus and before and after receiving the stimulus. Examples of the external stimulus include temperature change, light irradiation, electric field application, pH change, solute concentration change, solvent composition change and the like. A temperature change is preferable as the external stimulus. This is because it has little effect on cells and is easy to operate by simply controlling the ambient temperature.
  • a polymer gel whose shape and / or properties change in response to a temperature change is called a temperature-responsive polymer gel.
  • a temperature-responsive polymer gel whose volume changes in response to a temperature change in an aqueous solution
  • Hydrophilic polymer gels having an upper critical shared temperature (UCST) that swells at a known temperature are known.
  • the hydrophilic polymer gel having LCST becomes hydrophilic and swells at a temperature lower than the critical temperature, and conversely becomes hydrophobic and contracted at a temperature higher than the critical temperature.
  • a hydrophilic polymer gel having UCST becomes hydrophilic and swells on the higher temperature side than the critical temperature, and conversely becomes hydrophobic and contracted on the lower temperature side than the critical temperature.
  • both a hydrophilic polymer gel having LCST and a hydrophilic polymer gel having UCST can be used.
  • a hydrophilic polymer gel having UCST can be used.
  • a hydrophilic polymer gel having LCST can be used.
  • a hydrophilic polymer gel having LCST is preferable. This is because the general cell culture temperature is 37 ° C., and the cell aggregate can be detached from the polymer gel at room temperature (20 ° C. ⁇ 5 ° C.).
  • LCST can be determined by visual observation of two-phase separation. That is, the temperature at which the hydrophilic polymer gel is changed from transparent to white or from white to transparent is determined by immersing the hydrophilic polymer gel in an aqueous solution and gradually increasing or decreasing the temperature. Alternatively, a method can be used in which the size and weight of the gel are measured and the temperature at which the size or weight changes is obtained.
  • the hydrophilic polymer gel having LCST the following water-soluble monomer homopolymers or cross-linked copolymers can be used.
  • the water-soluble monomer include N-alkyl-substituted (meth) acrylamide and N, N-dialkyl-substituted (meth) acrylamide.
  • the alkyl group has 1 to 4 carbon atoms.
  • Specific examples of these water-soluble monomers include N-isopropylacrylamide (32 ° C), N-isopropylmethacrylamide (43 ° C), Nn-propylacrylamide (22 ° C), Nn-propylmethacrylamide (27 ° C).
  • N-cyclopropyl acrylamide 43 ° C.
  • N-cyclopropyl methacrylamide 60 ° C.
  • N-ethoxyethyl acrylamide 35 ° C.
  • N-ethoxyethyl methacrylamide 45 ° C.
  • N-ethyl acrylamide 72 ° C
  • N, N-diethylacrylamide 32 ° C
  • the temperature in said parenthesis shows LCST of a homopolymer.
  • LCST can be adjusted by copolymerizing these hydrophilic monomers and / or adjusting the type and concentration of the crosslinking agent.
  • LCST is preferably in the range of 37 ° C to 0 ° C. This is because if the temperature is lower than 0 ° C., the cell growth rate is extremely reduced or the cells are killed. Moreover, it is because it will become easy to peel at the time of culture
  • copolymer examples include those containing two or more of the above water-soluble monomers, and examples thereof include a combination of N-isopropylacrylamide and N, N-diethylacrylamide.
  • the copolymer may contain a water-soluble monomer such as acrylamide, acryloylmorpholine, N, N-dimethylaminopropyl acrylamide, etc., if necessary.
  • a water-soluble monomer such as acrylamide, acryloylmorpholine, N, N-dimethylaminopropyl acrylamide, etc., if necessary.
  • the hydrophilic polymer gel used in the present invention is obtained by polymerizing water-soluble monomers such as N-alkyl-substituted (meth) acrylamide and N, N-dialkyl-substituted (meth) acrylamide in the presence of a crosslinking agent and a polymerization initiator.
  • a crosslinking agent a bifunctional crosslinking agent or a trifunctional crosslinking agent can be used.
  • Bifunctional crosslinking agents include bifunctional acrylamides such as N, N-methylenebis (meth) acrylamide and N, N-propylenebis (meth) acrylamide, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, Mention may be made of alkylene glycol di (meth) acrylates such as polyethylene glycol di (meth) acrylate or polyalkylene glycol di (meth) acrylates.
  • Examples of the trifunctional crosslinking agent include triallyl cyanurate and triallyl isocyanurate.
  • the LCST, transparency and expandability of the hydrophilic polymer gel can be adjusted by changing the type and concentration of the crosslinking agent.
  • the LCST can be lowered. Therefore, the crosslinker concentration is adjusted so that the LCST is lower than the culture temperature.
  • the LCST can be adjusted by selecting a monomer species for copolymerizing the hydrophilic monomer. For example, LCST can be lowered by copolymerizing with a more hydrophobic monomer.
  • the transparency of the gel refers to the light transmittance at a wavelength of 500 nm at the cell culture temperature. Generally, when the crosslinker concentration is increased, the transparency is lowered. The transparency of the gel is not always essential, but the higher the light transmittance, the easier it is to observe the state of the cells during the cell culture period.
  • the light transmittance is 70% or more, preferably 90% or more.
  • the crosslinking agent imparting such transparency alkylene glycol di (meth) acrylate or polyalkylene glycol di (meth) acrylate is preferable.
  • the expansibility of a gel means the degree of expansion
  • the degree of swelling is preferably 1.1 or more. More preferably, it is 1.1-5.
  • the expansion of the gel includes anisotropic expansion and isotropic expansion, but isotropic expansion is preferable. This is because, in anisotropic expansion, only the specific direction of the gel expands, and as a result, the cells are easily damaged at the time of peeling.
  • Isotropic expansion can be represented by a change in gel length or a change in gel surface area.
  • a length-based expansion coefficient or a surface area-based expansion coefficient represented by the following formula is used for a rectangular sheet-like gel.
  • the length means the length of the rectangular sheet in the x-axis direction or the y-axis direction
  • the surface area means a two-dimensional plane defined by the length of the rectangular sheet in the x-axis direction and the length in the y-axis direction.
  • Length standard expansion coefficient (Gel length at temperature B / Gel length at temperature A) ⁇ 100 (%)
  • Surface area expansion coefficient (Gel surface area at temperature B / Gel surface area at temperature A) ⁇ 100 (%)
  • the length-based expansion coefficient is preferably 110% to 200%.
  • the surface area expansion coefficient is also the same, and is preferably 110% to 200%. In any case, if it exceeds 200%, the cells are easily damaged.
  • the length-based expansion coefficient or the surface area-based expansion coefficient becomes 110% or more over at least 1 minute after changing the temperature.
  • a crosslinking agent imparting isotropic expansibility alkylene glycol di (meth) acrylate or polyalkylene glycol di (meth) acrylate is preferable.
  • the hydrophilic polymer gel used in the present invention can be synthesized by known aqueous radical polymerization.
  • the polymerization is carried out in a polymerization vessel in the presence of a monomer such as the above hydrophilic monomer, the above crosslinking agent, and a polymerization initiator.
  • the polymerization container is preferably polymerized in a matrix described later having a predetermined three-dimensional structure.
  • the polymerization initiator those generally known as water-soluble radical initiators can be used.
  • Examples thereof include hydrogen peroxide, persulfates such as ammonium persulfate and potassium persulfate, and hydroperoxides such as t-butyl hydroperoxide.
  • concentration of the polymerization initiator is 0.01 to 10 mmol% with respect to the water-soluble monomer.
  • a particularly preferred hydrophilic polymer gel in the present invention is a homopolymer of N-isopropylacrylamide and polyethylene glycol dimethacrylate as a crosslinking agent, and the concentration of the crosslinking agent is that of N-isopropylacrylamide monomer. And 0.5 mmol% to 5 mmol%. This is because if the amount is less than 0.5 mmol%, the gel becomes cloudy, and if it exceeds 5 mmol%, the temperature responsiveness is lost.
  • the crosslinking agent concentration is 3.5 mmol%, this hydrophilic polymer gel has a light transmittance of 94% at a cell culture temperature of 37 ° C., and has high transparency. Further, when the temperature is lowered from 37 ° C. to 25 ° C., the cell aggregates are easily exfoliated due to isotropic expansion.
  • Polymer gel molding For the formation of the hydrophilic polymer gel, a method of polymerizing in a polymerization vessel having a three-dimensional structure or a method of cutting the polymerized gel can be used, but a method of polymerizing in a polymerization vessel having a three-dimensional structure is used. It is preferable to use it.
  • a resin matrix molded by a known resin molding method such as a casting method, a compression method, an injection molding method, an extrusion method, a three-dimensional printing method can be used. It is preferable to use a resin matrix produced by the original printing method.
  • the three-dimensional printing method is a method for producing a three-dimensional structure by continuously depositing or forming a thin layer material. For example, a liquid or powder monomer is sprayed onto a substrate, a print head is moved based on a stepped pattern, and a curing agent is sprayed by ink jet to produce a single layer, and the same procedure is repeated to laminate a plurality of layers in a predetermined three-dimensional manner. A resin matrix having a structure is produced. According to this three-dimensional printing method, even a matrix having a complicated shape can be manufactured with higher accuracy and lower cost than in the past. Therefore, it becomes possible to reproduce the fine form of the transplanted site such as a defective site or to accurately give an arbitrary shape to the three-dimensional cell aggregate.
  • thermosetting resin or photocurable resin used in the three-dimensional printing method can be used.
  • the cells are cultured by seeding the cells on a carrier made of a molded body of a hydrophilic polymer gel and culturing the cells in a predetermined medium for a predetermined period.
  • a carrier made of a molded body of a hydrophilic polymer gel
  • the cell concentration is 0.5 ⁇ 10 6 to 2 ⁇ 10 6 cells / ml, preferably 1 ⁇ 10 6 cells / ml.
  • a culture solution of ⁇ 2 ⁇ 10 6 cells / ml is seeded on a three-dimensional gel carrier of polymer gel.
  • the medium and culture conditions can be selected according to the type of cells to be cultured.
  • Cultured cells are detached as a three-dimensional cell aggregate by changing the volume of the hydrophilic polymer gel as a carrier.
  • a hydrophilic polymer gel having a lower critical solution temperature (LCST) that shrinks at a critical temperature (Tc) or higher is used
  • a hydrophilic polymer gel having a Tc lower than the culture temperature for example, 37 ° C.
  • the temperature of the hydrophilic polymer gel is lowered to a temperature lower than the LCST after completion of the culture, the hydrophilic polymer gel swells and the cultured cells are detached accordingly.
  • LCST poly (N-isopropylacrylamide) gel
  • the gel is cultured at 37 ° C., and the gel temperature is lower than LCST, that is, 32 ° C. or lower. By lowering, the cultured cells are detached from the gel.
  • Cells that can be cultured using the present invention include stem cells such as ES cells (Embryonic stem cells), iPS cells (induced pluripotent stem cells), mesenchymal stem cells, various epithelial and mesenchymal somatic cells, All animal cells, such as tumor cells. In addition to animal cells, all cells can be cultured regardless of eukaryotic or prokaryotic, such as insects, plants or bacteria.
  • stem cells such as ES cells (Embryonic stem cells), iPS cells (induced pluripotent stem cells), mesenchymal stem cells, various epithelial and mesenchymal somatic cells.
  • All animal cells such as tumor cells.
  • all cells can be cultured regardless of eukaryotic or prokaryotic, such as insects, plants or bacteria.
  • an organic substance that promotes cell adhesion is a substance that is involved in adhesion between cells or between cells and extracellular matrix, and includes laminin, collagen, fibronectin, gelatin, and various peptides such as RGD, IKVAV, and YIGSR.
  • fibronectin or gelatin is preferred.
  • the concentration of fibronectin is 0.5 ⁇ g / ml or more, preferably 1.0 ⁇ g / ml or more.
  • a three-dimensional cell aggregate obtained by using the production method of the present invention is cultured using a molded article of a hydrophilic polymer gel having a three-dimensional structure and having a variable volume as a template, and the three-dimensional structure is imparted by the template. It consists of an aggregate of cells.
  • the size and thickness of the three-dimensional cell aggregate of the present invention are not particularly limited, and can be appropriately selected depending on the application.
  • the following uses are considered for the three-dimensional cell aggregate of the present invention.
  • Transplant material for tissue regeneration Based on one or more types of cells, it can be used as a transplant material reflecting the size and form of the defect site. It can also be used as a transplant material that induces tissue regeneration.
  • Artificial embryo production material based on iPS cells and ES cells There is a possibility that an artificial early embryo of a specific size can be produced based on a certain number of cells.
  • Tool for in vitro examination of tissue morphogenesis process Tissue morphogenesis by using not only early embryos using ES cells, but also cells that have undergone differentiation to some extent and using them to produce cell aggregates It can be used as a study tool.
  • Synthesis Example 1 (Preparation of poly (N-isopropylacrylamide) gel) N-isopropylacrylamide (NIPAAm) (manufactured by Kojin Co., Ltd.) was recrystallized from hexane and used. Polyethylene glycol dimethacrylate (PEG-DMA) (manufactured by Sigma) was used as a cross-linking agent.
  • NIPAAm N-isopropylacrylamide
  • PEG-DMA Polyethylene glycol dimethacrylate
  • NIPAAm 0.79 g of NIPAAm was dissolved in 10 ml of ultrapure water, then 16 mg of ammonium persulfate (APS) (manufactured by Nacalai Tesque) as an initiator (1 mol% based on the total monomers) and N, N, N, N-tetramethylethylenediamine 10 ⁇ l of (TEMED) (manufactured by Nacalai) was added.
  • the mixed solution was poured into a glass mold (size: 100 ⁇ 100 ⁇ 1 mm) and left in an incubator at 37 ° C. for 2 hours.
  • the polymerized gel was removed from the mold and washed with distilled water for 72 hours and then with alcohol (70% solution) for 48 hours.
  • the resulting gel had an LCST of 32 ° C.
  • Synthesis Example 2 A gel was prepared in the same manner as in Synthesis Example 1 except that N, N′-bisacrylamide (MBAA) (manufactured by Sigma) was used as the crosslinking agent instead of PEG-DMA.
  • the resulting gel had an LCST of 32 ° C.
  • the contact angle of bubbles was measured by an underwater gas method using a contact angle meter (Drop Master 500 manufactured by Kyowa Interface Science Co., Ltd.). In the underwater gas method, 10 ⁇ l of air was contacted from below while the gel was immersed in water, and the contact angle after 30 seconds was measured at room temperature.
  • the gel synthesized in Synthesis Example 1 was used as the gel.
  • Human organic fibronectin (FN) (0-2 ⁇ g / ml, manufactured by BD Biosciences) or gelatin (1% by weight / ml, manufactured by Wako Pure Chemical Industries) was used as the organic substance that promotes cell adhesion.
  • the gel was immersed in a phosphate buffered saline (PBS) solution in which FN or gelatin was dissolved, and incubated for 6 hours.
  • PBS phosphate buffered saline
  • DMEM Dulbecco's modified Eagle's medium
  • the number of cells was measured using a hemocytometer.
  • the adhered cells and the cell sheet detached from the gel were observed using an optical microscope (Nikon TE2000, CoolSNAP) equipped with a CCD camera.
  • both the bottom surface of the obtained cell sheet and the gel surface from which the cell sheet was peeled off were used using anti-FN and Sigma fluorescent immunostaining of FN. Went about. Digital images of gel templates and cell aggregates were taken and analyzed using image analysis software (Image J, NIH).
  • the adhesion surface of the cell sheet with the gel was observed with a scanning electron microscope (JEOL 6350, manufactured by JEOL Ltd.).
  • the gel using PEG-DMA as a crosslinking agent was transparent at 37 ° C. and had a light transmittance of 94%.
  • the gel using MBAA changed from transparent to white when the temperature was raised from 15 ° C to 37 ° C.
  • FIG. 2A is a graph showing the relationship between the temperature and the degree of swelling of a gel using PEG-DMA as a crosslinking agent, and the degree of swelling is calculated based on the gel weight at 37 ° C. From this graph, the degree of swelling at 25 ° C. was about 4.
  • FIG. 2B is a graph showing the relationship between the length standard expansion coefficient of gel using PEG-DMA as a crosslinking agent and time at a temperature of 25 ° C.
  • the gel-length expansion coefficient was about 110% in 1 minute, 120% in 10 minutes, about 130% in 20 minutes, and became almost constant after about 60 minutes, and was about 137%.
  • FIG. 2C is an appearance photograph showing the reversible expansion / contraction state of the gel using PEG-DMA as a cross-linking agent, with the left side at 25 ° C. and the right side at 37 ° C.
  • the surface area standard expansion coefficient accompanying the temperature change from 37 ° C. to 25 ° C. calculated from this photograph was about 140%.
  • FIG. 2D is a graph showing the contact angle of bubbles at 25 ° C. and 37 ° C. of a gel using PEG-DMA as a cross-linking agent.
  • the contact angle did not change between 25 ° C. and 37 ° C. and had a large angle of about 150 degrees.
  • the contact angle indicates the degree of hydrophilicity / hydrophobicity of the material surface, and this result indicates that there is no significant change in the hydrophilicity / hydrophobicity of the gel surface between 25 ° C and 37 ° C.
  • the gel using PEG-DMA as a cross-linking agent shows a large expansion coefficient when the temperature is lowered from 37 ° C. to 25 ° C., but there is no change in the hydrophilicity / hydrophobicity of the gel surface. It was.
  • FIG. 3 is an optical microscope image showing the state of cells on the gel surface, A is a control (without applying organic substance that promotes cell adhesion), and B is gelatin. (1 wt%) applied, C applied FN at a concentration of 1 ⁇ g / ml, and D applied FN 2 ⁇ g / ml.
  • FIG. 4 is a graph showing the relationship between time and the number of cells, where ⁇ indicates A, ⁇ indicates B, ⁇ indicates C, and ⁇ indicates the result for D.
  • FN has a greater effect of increasing the number of cells than gelatin.
  • a sufficient effect can be obtained at a concentration of 1 ⁇ g / ml or more. Therefore, a gel coated with FN at a concentration of 1 ⁇ g / ml was used in the subsequent experiments.
  • Cell sheet A cell sheet was prepared using a gel using PEG-DMA as a crosslinking agent as a carrier. When the seeded cells were confluent on the gel and the gel temperature was lowered from 37 ° C. to 25 ° C., the cell sheet could be detached from the gel without losing its shape.
  • Experimental Example 2 (Production of three-dimensional cell aggregate) A gel serving as a template for preparing a three-dimensional cell aggregate was prepared using a resin mold.
  • the resin mold was designed using graphic modeling software (FreeForm, Sensable, MA), and produced using a three-dimensional printing system (Eden, Objet, Israel).
  • FIG. 5A is a model diagram of a mother die produced using the graphic modeling software described above, and shows a mother die having a plurality of parallel ridges and a mother die having a plurality of hemispherical protrusions.
  • FIG. 5B is a photograph showing the appearance of a matrix made of a photocurable resin produced using the above-described three-dimensional printing system, with the upper side having a plurality of parallel protrusions and the lower side having a plurality of hemispheres. A matrix having a convex portion is shown.
  • FIG. 5C is a photograph showing the appearance of a gel molded body (hereinafter also referred to as a gel mold) which is polymerized in the resin mold and taken out from the resin mold. It has been transcribed. The upper side shows a mold having a plurality of parallel grooves, and the lower side shows a mold having a plurality of hemispherical recesses.
  • a gel molded body hereinafter also
  • Osteoblast MC3T3-E1 is seeded at a cell concentration of 1 ⁇ 10 6 cells / ml in a gel template having a plurality of parallel grooves shown in FIG. 5C, cultured at 37 ° C. for 48 hours, and the temperature is lowered to 25 ° C.
  • the aggregate was peeled from the gel mold to obtain a rod-shaped cell aggregate.
  • the gel mold used was one with and without FN coating.
  • FIG. 5D is a photograph showing the appearance of a gel template containing cultured cells.
  • FIG. 5E is a photograph showing the appearance of the cell aggregate taken out from the gel mold, in which the upper side shows a rod-like shape and the lower side shows hemispherical cell aggregates of various sizes.
  • FIG. 5G is a graph showing changes in the length of rod-shaped cell aggregates when FN is applied to a gel template and when FN is not applied (“length (%)” in the figure indicates the gel template). Represents the ratio of the length of rod-shaped cell aggregate to the length).
  • the length of the rod-shaped cell aggregate was 91% of the length of the groove of the gel template, whereas it was 65% when FN was not applied. From this, it was found that in order to produce a cell aggregate in accordance with the shape of the gel template, it is preferable to apply FN to the gel template.
  • Experimental Examples 3 to 5 are examples of the production of a transplant material for tissue regeneration, the production of an artificial tumor model, and the production of an artificial periosteum model performed using the three-dimensional cell assembly of the present invention.
  • the cell sheet could not be produced.
  • Experimental Example 3 (Endochondral ossification using 3D cell aggregate) As a gel used as a template for producing a three-dimensional cell aggregate, the gel of Synthesis Example 1 was used, and a resin mold was used in the same manner as in Experimental Example 2. Using a resin mold having a plurality of hemispherical convex portions, a gel having 20 hemispherical concave portions having a diameter of 1.5 mm was prepared, and bone marrow mesenchymal stem cells (BALB / c mouse were used as single cells in each concave portion. 20,000) were seeded and incubated.
  • a resin mold having a plurality of hemispherical convex portions a gel having 20 hemispherical concave portions having a diameter of 1.5 mm was prepared, and bone marrow mesenchymal stem cells (BALB / c mouse were used as single cells in each concave portion. 20,000) were seeded and incubated.
  • the temperature was lowered from 37 ° C. to 25 ° C. to peel off the gel and collect hemispherical three-dimensional cell aggregates.
  • the collected three-dimensional cell aggregate was transferred to a culture dish, and the dish was placed on a seesaw shaker, and shaking culture was continued for 50 days.
  • An osteoinduction medium was used as the medium.
  • FIG. 6 is a photograph showing the differentiation state of chondrocytes, and the chondrocyte differentiation could be confirmed by the fact that a deeply stained image with toluidine blue was observed in the cell layer a little inside from the outermost side.
  • FIG. 7 is a photograph showing the state of calcification, the left photograph shows the internal image of the cell aggregate 20 days after the start of the shaking culture, and the right photograph shows the internal image of the cell aggregate 50 days after the start of the shaking culture. Show. A dark brown stained image by von Kossa staining was observed in the cell aggregate and the inner cell layer after 50 days of culture. Using the three-dimensional cell aggregate of the present invention, endochondral ossification could be reproduced in vitro.
  • Experimental example 4 artificial tumor model using three-dimensional cell aggregates
  • the gel used as a template for producing a three-dimensional cell aggregate was prepared by the same method as in Experimental Example 3. Twenty thousand squamous cell carcinoma cells (A549) were seeded in each concave portion of a gel having 20 hemispherical concave portions having a diameter of 1.5 mm and incubated.
  • the temperature was lowered from 37 ° C. to 25 ° C. to peel off the gel and collect hemispherical three-dimensional cell aggregates.
  • the collected three-dimensional cell aggregate was transferred to a culture dish, and the dish was placed on a seesaw shaker, and shaking culture was continued for 10 days.
  • a DMEM medium containing 10% FBS was used as the medium.
  • HIF1 ⁇ which is an indicator of hypoxia
  • VEGF vascular endothelial inducer
  • FIG. 8 shows a fluorescent immunostaining image in which cells in the outer layer of the cell assembly express HIF1 ⁇ .
  • FIG. 9 shows a fluorescent immunostaining image in which cells in the outer layer of the cell assembly express VEGF. This expression was consistent with the expression localization of HIF1 ⁇ in FIG.
  • Experimental example 5 artificial periosteal model using a three-dimensional cell aggregate
  • the gel of Synthesis Example 1 was used, and a resin mold was used in the same manner as in Experimental Example 2.
  • a resin mold having a rectangular parallelepiped convex portion a gel having a 15 ⁇ 15 ⁇ 1 mm (vertical ⁇ horizontal ⁇ thickness) concave portion was prepared, and a bone marrow mesenchymal stem cell (BALB / c mouse) was formed in this concave portion. 2 million seeds were seeded.
  • the temperature was lowered from 37 ° C. to 25 ° C. to peel the gel, and the sheet-like three-dimensional cell aggregate was collected.
  • a bone filling material (zirconia / apatite complex or collagen sponge) is added to a cylindrical defect (diameter 4 mm) artificially prepared on the rat skull, and then the collected sheet-like three-dimensional cell aggregate is pressure-welded. The skin was sutured.
  • FIG. 10 shows an HE-stained image of a non-decalcified section.
  • the arrow part shows the transplanted sheet-like three-dimensional cell aggregate.
  • the formation of new bone around the bone filling material located inside the sheet was confirmed, suggesting that the transplanted sheet-like three-dimensional cell aggregate blocks the invasion of epithelial tissues and the like, and effectively works for bone regeneration. .
  • the present invention it is possible to easily produce a three-dimensional cell aggregate having an arbitrary shape that has been difficult to produce.
  • the three-dimensional cell aggregate obtained by the present invention not only provides a new tissue regeneration tool, but can also be used as a tool for pseudo-tumor tissue and bacterial infection model construction and drug screening. .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

L'invention concerne un procédé permettant de créer un agrégat cellulaire tridimensionnel. Un article tridimensionnel moulé à partir d'un gel de polymère hydrophile pouvant changer de volume est utilisé comme support. Le support est inoculé avec des cellules et les cellules sont cultivées. Le volume du support change, et les cellules cultivées ainsi dotées d'une structure tridimensionnelle sont détachées comme agrégat cellulaire tridimensionnel. Il est donc possible de créer un agrégat cellulaire tridimensionnel de toute forme sans recourir à une procédure complexe.
PCT/JP2011/054573 2010-03-08 2011-03-01 Procédé de création d'un agrégat cellulaire tridimensionnel, d'un support de gel tridimensionnel pour la culture cellulaire utilisé dans ce procédé, et agrégat cellulaire tridimensionnel Ceased WO2011111562A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2010-050926 2010-03-08
JP2010050926A JP2013116045A (ja) 2010-03-08 2010-03-08 三次元細胞集合体の作製方法およびそれに用いる細胞培養用三次元ゲル担体並びに三次元細胞集合体

Publications (1)

Publication Number Publication Date
WO2011111562A1 true WO2011111562A1 (fr) 2011-09-15

Family

ID=44563369

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2011/054573 Ceased WO2011111562A1 (fr) 2010-03-08 2011-03-01 Procédé de création d'un agrégat cellulaire tridimensionnel, d'un support de gel tridimensionnel pour la culture cellulaire utilisé dans ce procédé, et agrégat cellulaire tridimensionnel

Country Status (2)

Country Link
JP (1) JP2013116045A (fr)
WO (1) WO2011111562A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103801697A (zh) * 2014-02-11 2014-05-21 北京科技大学 一种金属浆料3d打印无模凝胶成形方法
JP2014097031A (ja) * 2012-11-15 2014-05-29 Dainippon Printing Co Ltd 温度応答性を有する細胞培養基材の製造方法
JP5946046B2 (ja) * 2012-12-27 2016-07-05 新田ゼラチン株式会社 ヒト角膜内皮細胞シート
CN107164305A (zh) * 2017-04-27 2017-09-15 西安交通大学 一种体外类脑组织及其构建方法
CN109762802A (zh) * 2019-01-28 2019-05-17 北京华龛生物科技有限公司 一种细胞载体颗粒聚集体及其制备方法
WO2021014710A1 (fr) * 2019-07-22 2021-01-28 横河電機株式会社 Mécanisme de récupération de composant, récipient de récupération de composant, kit de récupération de composant, système de récupération de composant, récipient de culture, système de culture et procédé de production de cellules
RU229942U1 (ru) * 2024-09-22 2024-11-06 Андрей Алексеевич Мошин Аппарат для объёмного культивирования клеток

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11097532B2 (en) 2016-02-01 2021-08-24 Ricoh Company, Ltd. Method for making mold, method for molding model material, and mold making apparatus
KR102228619B1 (ko) * 2019-11-25 2021-03-17 경희대학교 산학협력단 히알루론산 및 폴리에틸렌글리콜을 포함하여 제조된 세포 시트 및 이의 제조방법

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008011766A (ja) * 2006-07-05 2008-01-24 Mitsuo Okano 細胞培養支持体
JP2008126459A (ja) * 2006-11-20 2008-06-05 Kanagawa Acad Of Sci & Technol ゲルの製造方法及びそのための装置
JP2010017128A (ja) * 2008-07-10 2010-01-28 Toyota Central R&D Labs Inc 培養細胞のハンドリング体、その製造方法及びその利用
JP2010057439A (ja) * 2008-09-05 2010-03-18 Konica Minolta Holdings Inc 細胞培養支持体とその製造方法並びに細胞培養方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008011766A (ja) * 2006-07-05 2008-01-24 Mitsuo Okano 細胞培養支持体
JP2008126459A (ja) * 2006-11-20 2008-06-05 Kanagawa Acad Of Sci & Technol ゲルの製造方法及びそのための装置
JP2010017128A (ja) * 2008-07-10 2010-01-28 Toyota Central R&D Labs Inc 培養細胞のハンドリング体、その製造方法及びその利用
JP2010057439A (ja) * 2008-09-05 2010-03-18 Konica Minolta Holdings Inc 細胞培養支持体とその製造方法並びに細胞培養方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JUN-ICHI SASAKI ET AL.: "Fabrication of three- dimensional cell constructs using temperature- responsive hydrogel.", TISSUE ENGINEERING PART A, vol. 16, no. 8, November 2010 (2010-11-01), pages 2497 - 2504 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014097031A (ja) * 2012-11-15 2014-05-29 Dainippon Printing Co Ltd 温度応答性を有する細胞培養基材の製造方法
JP5946046B2 (ja) * 2012-12-27 2016-07-05 新田ゼラチン株式会社 ヒト角膜内皮細胞シート
CN103801697A (zh) * 2014-02-11 2014-05-21 北京科技大学 一种金属浆料3d打印无模凝胶成形方法
CN107164305A (zh) * 2017-04-27 2017-09-15 西安交通大学 一种体外类脑组织及其构建方法
CN109762802A (zh) * 2019-01-28 2019-05-17 北京华龛生物科技有限公司 一种细胞载体颗粒聚集体及其制备方法
WO2021014710A1 (fr) * 2019-07-22 2021-01-28 横河電機株式会社 Mécanisme de récupération de composant, récipient de récupération de composant, kit de récupération de composant, système de récupération de composant, récipient de culture, système de culture et procédé de production de cellules
JP2021016360A (ja) * 2019-07-22 2021-02-15 横河電機株式会社 成分回収機構、成分回収容器、成分回収キット、成分回収システム、培養用容器、培養システム、および細胞の製造方法
JP7044098B2 (ja) 2019-07-22 2022-03-30 横河電機株式会社 成分回収機構、成分回収容器、成分回収キット、成分回収システム、培養用容器、培養システム、および細胞の製造方法
US12454672B2 (en) 2019-07-22 2025-10-28 Yokogawa Electric Corporation Component recovery mechanism, component recovery container, component recovery kit, component recovery system, culture container, culture system, and method of producing cells
RU229942U1 (ru) * 2024-09-22 2024-11-06 Андрей Алексеевич Мошин Аппарат для объёмного культивирования клеток

Also Published As

Publication number Publication date
JP2013116045A (ja) 2013-06-13

Similar Documents

Publication Publication Date Title
WO2011111562A1 (fr) Procédé de création d'un agrégat cellulaire tridimensionnel, d'un support de gel tridimensionnel pour la culture cellulaire utilisé dans ce procédé, et agrégat cellulaire tridimensionnel
JP2020036621A (ja) 細胞培養支持体、細胞培養装置、細胞培養キット、及び細胞シート
Seo et al. Interconnectable dynamic compression bioreactors for combinatorial screening of cell mechanobiology in three dimensions
US9908972B2 (en) Photodegradable cross-linking agent, photodegradable gel, cell culture instrument, cell arrangement-sorting apparatus, cell arrangement method, cell sorting method, tissue forming method, and tissue
JP2010161952A (ja) 生体組織の作製方法
JP5396803B2 (ja) 細胞培養基材及び細胞培養方法
Parke-Houben et al. Interpenetrating polymer network hydrogel scaffolds for artificial cornea periphery
CN107075443B (zh) 三维细胞培养系统和使用该系统的细胞培养方法
WO2011163551A2 (fr) Échafaudage tissulaire biomimétique et ses procédés de fabrication et d'utilisation
JP2010516273A (ja) 培養細胞を処理する方法
Van Damme et al. Indirect versus direct 3D printing of hydrogel scaffolds for adipose tissue regeneration
Tejavibulya et al. Directed self-assembly of large scaffold-free multi-cellular honeycomb structures
Kim et al. Thermoresponsive terpolymeric films applicable for osteoblastic cell growth and noninvasive cell sheet harvesting
JP2017147951A (ja) 細胞培養方法及び培養組織
Kim et al. Successive grafting of PHEMA and PIPAAm onto cell culture surface enables rapid cell sheet recovery
Li et al. A review on thermoresponsive cell culture systems based on poly (N-isopropylacrylamide) and derivatives
JP7416185B2 (ja) 立体的細胞構造体の製造方法
Ahmad et al. Coaxial bioprinting of a stentable and endothelialized human coronary artery-sized in vitro model
JP2010046012A (ja) 細胞培養基材及び細胞培養方法
JP5713086B2 (ja) 生体組織の作製方法
Liu et al. Rapid aqueous photo‐polymerization route to polymer and polymer‐composite hydrogel 3D inverted colloidal crystal scaffolds
US20180127700A1 (en) Cell Culture Substrate for Rapid Release and Re-Plating
JPH03266980A (ja) 細胞培養用基材およびそれを用いた細胞集合体の製造方法
JP6229503B2 (ja) 温度応答性を有する細胞培養基材およびその製造方法
JP6953863B2 (ja) 細胞シートの剥離性の調整方法、細胞シートの製造方法、及び細胞培養容器の製造方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11753222

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11753222

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP