WO2006093207A1 - Matériau de base pour la régulation de la différenciation/prolifération de cellules - Google Patents

Matériau de base pour la régulation de la différenciation/prolifération de cellules Download PDF

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Publication number
WO2006093207A1
WO2006093207A1 PCT/JP2006/303909 JP2006303909W WO2006093207A1 WO 2006093207 A1 WO2006093207 A1 WO 2006093207A1 JP 2006303909 W JP2006303909 W JP 2006303909W WO 2006093207 A1 WO2006093207 A1 WO 2006093207A1
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Prior art keywords
culture substrate
substrate according
stem cells
cells
culture
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PCT/JP2006/303909
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English (en)
Japanese (ja)
Inventor
Masaru Tanaka
Akinori Tsuruma
Sada-Aki Yamamoto
Masatsugu Shimomura
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National University Corporation Hokkaido University
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Priority to JP2007505992A priority Critical patent/JP4956753B2/ja
Publication of WO2006093207A1 publication Critical patent/WO2006093207A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • 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
    • C12M25/02—Membranes; Filters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers

Definitions

  • the present invention relates to a structure capable of freely controlling the morphology of a cell. More specifically, the present invention relates to a structure capable of freely manipulating stem cell sorting or proliferation. It relates to the structure. Background art
  • ES cells embryonic stem cells
  • ES cells embryonic stem cells
  • ES cells that have self-renewal ability (self-amplification ability) and pluripotency (ability to differentiate into all cell types that form an individual) have these ability.
  • regenerative medicine for example, medicine in which desired cells and Z or tissue are produced as needed and transplanted into a living body is expected.
  • stem cells following the establishment of embryonic stem cells, which have mainly been studied for hematopoietic stem cells, stem cells of each cell lineage (eg, liver, muscle, skin, nerve, etc.) have also been identified to date. ing. It is considered that development of a technique for modifying these stem cells at the gene level and modifying their functions as necessary will lead to the development of treatments for intractable diseases (for example, cancer and degenerative diseases). In fact, hematopoietic stem cells have already been applied to bone marrow transplantation, and mouse embryonic stem cells are used in gene targeting. Sarasuko, recently isolated human embryonic stem cells are expected to be applied to organ formation via transplantation, and normal tissue stem cells can be used for cancer therapy or regenerative medicine, or gene therapy targeting tissue stem cells. Application is also expected!
  • somatic stem cells ie, tissue stem cells such as hematopoietic stem cells and neural stem cells
  • organs It was thought to have only regenerative capacity.
  • Non-Patent Document 1 hematopoietic stem cells can be amplified in vitro by using neural stem cells that can be cultured and passaged .
  • the scaffold is an artificial object, it can induce cell adhesion, proliferation and Z or sorting based on its three-dimensional structure, and the scaffold incorporating the cell It has been found that tissue can be reconstructed by transplanting the organism into a living body.
  • tissue can be reconstructed by transplanting the organism into a living body.
  • it is possible to construct artificial neural circuits by controlling the adhesion form of neurites and the extension of neurites by using various micropatterned substrates produced by microfabrication technology. It has been studied and is expected to be applied to nerve regeneration.
  • micropattern technology requires a very high level of technology, has many problems such as high mass production and high cost.
  • the present inventor is able to prepare economically by combining a biodegradable polymer and an amphiphilic polymer in an appropriate ratio, and has a self-supporting and structurally stable structure. And a cell culture substrate using the structure is completed (for example, see Patent Documents 1 and 2).
  • Patent Document 1 Japanese Patent Laid-Open No. 2002-347107 (published on December 4, 2002)
  • Patent Document 2 JP 2002-335949 A (published on November 26, 2002)
  • Non-patent literature l Bjornson, C. R. et al., Science 283: 534-537 (1999).
  • stem cells As described above, many attempts have been made to apply stem cells to gene therapy, organ transplantation, bone marrow transplantation, cancer therapy, or regenerative medicine.
  • a major challenge in research using stem cells is that no stem cell-specific markers have been discovered that have very few stem cells. For this reason, it is difficult to purify stem cells, and no technology has been developed for self-proliferation without differentiation of stem cells without adding growth factors.
  • a technique for controlling cell differentiation without adding a differentiation-inducing factor has not been developed.
  • the present invention has been made in view of the above-described problems, and an object of the present invention is to provide a structure capable of freely controlling the morphology of cells and to automate the differentiation and proliferation of stem cells. It is to provide a structure that can be freely operated.
  • the culture substrate according to the present invention is characterized in that stem cells are proliferated without differentiation.
  • the culture substrate according to the present invention preferably includes a thin film having a film thickness ranging from 0.01 to LOO m.
  • the culture substrate according to the present invention comprises a plurality of the above thin films.
  • the culture substrate according to the present invention is preferably made of sallow.
  • the scab comprises a biodegradable polymer.
  • the scab further contains an amphiphilic polymer.
  • the above-mentioned coconut resin comprises a biodegradable polymer and an amphiphilic polymer!
  • the biodegradable polymer is selected from the group consisting of polylactic acid, poly ( ⁇ -force prolatatone), and poly (glycolic acid-lactic acid) copolymer. It is preferable that
  • the amphiphilic polymer has a dodecyl group as a hydrophobic side chain and has a ratato group or a carboxyl group as a hydrophilic side chain.
  • Amphiphilic resin having acrylamide as main chain skeleton; Polyethylene glycol copolymer; and polyion complex of ionic polymer and long chain alkyl ammonium salt. Preferably selected from the group.
  • the culture substrate according to the present invention preferably has a plurality of pores.
  • the average pore diameter is preferably 0.1 to 20 ⁇ m.
  • the coefficient of variation in pore diameter is preferably 30% or less.
  • the width between the pores is preferably 0.01 to 7 ⁇ m.
  • the plurality of holes are arranged in a Harkham-like manner.
  • each hole penetrates the culture substrate according to the present invention.
  • the holes communicate with each other!
  • the stem cells are preferably selected from the group consisting of neural stem cells, hematopoietic stem cells, mesenchymal stem cells, somatic stem cells, and embryonic stem cells U, .
  • the culture substrate according to the present invention is characterized by differentiating stem cells.
  • the culture substrate according to the present invention preferably comprises a thin film having a film thickness in the range of 0.01 to LOO m.
  • the culture substrate according to the present invention comprises a plurality of the above thin films.
  • the culture substrate according to the present invention comprises sallow.
  • the coconut resin contains a biodegradable polymer.
  • the scab further contains an amphiphilic polymer.
  • the above-mentioned coconut resin comprises a biodegradable polymer and an amphiphilic polymer!
  • the biodegradable polymer is selected from the group consisting of polylactic acid, poly ( ⁇ -force prolatatone), and poly (glycolic acid-lactic acid) copolymer. It is preferable that
  • the amphiphilic polymer has a dodecyl group as a hydrophobic side chain and a ratato group or a carboxyl group as a hydrophilic side chain.
  • a dodecyl group as a hydrophobic side chain and a ratato group or a carboxyl group as a hydrophilic side chain.
  • an amphiphilic resin having an acrylamide polymer as a main chain skeleton; a polyethylene glycol copolymer; and a polyion complexing force between an ionic polymer and a long-chain alkyl ammonium salt Preferred to be selected.
  • the culture substrate according to the present invention preferably has a plurality of pores.
  • the culture substrate according to the present invention preferably has an average pore diameter of 0.1 to 20 ⁇ m.
  • the pore diameter variation coefficient is preferably 30% or less.
  • the width between the pores is 0.01 to 7 ⁇ m. preferable.
  • the plurality of holes are arranged in a Herkam-like manner.
  • each of the holes penetrates the culture substrate according to the present invention.
  • the holes communicate with each other.
  • the stem cells are preferably selected from the group consisting of neural stem cells, hematopoietic stem cells, mesenchymal stem cells, somatic stem cells and embryonic stem cells. .
  • FIG. 1 is a diagram showing the shape of a porous film according to one embodiment of the present invention, and the pore diameter, trunk diameter, and porosity measured in the film.
  • Fig. 2a shows the results of seeding cells prepared from mouse fetal cerebral cortical tissue on PCL flat membranes and performing immunochemical staining for Nestin 4 hours later (left). The right figure shows the fluorescent image of Phalloidin performed at the same time.
  • FIG. 2b Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a PCL flat membrane, and immunochemical staining for BudU was performed 4 hours later (left). The right figure shows the fluorescence image of Phalloidin performed at the same time.
  • FIG. 3a Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a porous film having a pore diameter of 3 ⁇ m, and immunochemical staining for Nestin was performed 4 hours later (left figure). The right figure shows the fluorescence image of Phalloidin performed simultaneously.
  • FIG. 3b Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a porous film having a pore size of 3 ⁇ m, and immunochemical staining for BudU was performed 4 hours later (left). The right figure shows the fluorescence image of Phalloidin performed simultaneously.
  • FIG. 4a is a diagram showing the results of observing the morphology of cells after 5 days in culture with a scanning electron microscope after seeding cells prepared from mouse fetal cerebral cortex tissue on a PCL flat membrane.
  • FIG. 4b Mouse fetal cerebral cortex tissue strength The prepared cells were placed in PCL porous membranes with a pore size of 2-3 ⁇ m.
  • FIG. 3 is a diagram showing the results of observing with a scanning electron microscope the morphology of cells seeded on rum and cultured after 5 days.
  • FIG. 5c is a schematic diagram of the cell morphology shown in b.
  • FIG. 6a Mouse embryonic cerebral cortex tissue strength The prepared cells were seeded on a 3 ⁇ m pore PCL porous film, and immunochemical staining with j8-tubulin III was observed with a confocal laser microscope after 5 days in culture.
  • FIG. 6a Mouse embryonic cerebral cortex tissue strength The prepared cells were seeded on a 3 ⁇ m pore PCL porous film, and immunochemical staining with j8-tubulin III was observed with a confocal laser microscope after 5 days in culture.
  • FIG. 6b Mouse embryonic cerebral cortex tissue strength The prepared cells were seeded on a PCL porous film with a pore size of 3 ⁇ m, and the morphology of the cells after 5 days in culture was observed with a scanning electron microscope. .
  • FIG. 6c is a schematic diagram of the cell morphology shown in b.
  • FIG. 7c is a schematic diagram of the cell morphology shown in b.
  • FIG. 8a Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a PCL porous film with a pore size of 8 ⁇ m, and immunochemical staining with j8-tubulin III was observed with a confocal laser microscope after 5 days in culture.
  • FIG. 8a Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a PCL porous film with a pore size of 8 ⁇ m, and immunochemical staining with j8-tubulin III was observed with a confocal laser microscope after 5 days in culture.
  • FIG. 8b Mouse fetal cerebral cortex tissue strength is a diagram showing the results of seeding the prepared cells on a PCL porous film with a pore size of 8 ⁇ m and observing the morphology of the cells after 5 days of culture with a scanning electron microscope. .
  • FIG. 8c is a schematic diagram of the cell morphology shown in b.
  • FIG. 9a Cells seeded with mouse embryonic cerebral cortical tissue force were seeded on PCL porous film with a pore size of 10 ⁇ m, and immunochemical staining for j8-tubulin III after 5 days in culture was observed with a confocal laser microscope.
  • FIG. 9a Cells seeded with mouse embryonic cerebral cortical tissue force were seeded on PCL porous film with a pore size of 10 ⁇ m, and immunochemical staining for j8-tubulin III after 5 days in culture was observed with a confocal laser microscope.
  • FIG. 9c is a schematic diagram of the cell morphology shown in b.
  • FIG. 12a Mouse embryonic cerebral cortex tissue strength. The prepared cells were seeded on a PCL porous film with a pore size of 3 ⁇ m, and immunochemical staining for BrdU after 3 days in culture was observed with a confocal laser microscope. is there.
  • FIG. 12b Mouse embryonic cerebral cortex tissue strength. The prepared cells were seeded on a PCL porous film with a pore size of 3 ⁇ m, and immunochemical staining for BrdU after 7 days in culture was observed with a confocal laser microscope. is there.
  • FIG. 13a Mouse embryonic cerebral cortex tissue strength Prepared cells were seeded on a 3 ⁇ m pore PCL porous film, and immunochemical staining for Nestin after 5 days in culture was performed using a confocal laser microscope. It is a figure which shows the result observed in (left figure). The right figure shows the fluorescence image of Phalloidin performed simultaneously.
  • FIG. 13b Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a PCL porous film with a pore size of 3 ⁇ m, and immunochemical staining for Nestin after 7 days in culture was observed with a confocal laser microscope. Yes (left). The right figure shows the fluorescence image of Phalloidin performed simultaneously.
  • FIG. 13c Mouse embryonic cerebral cortical tissue strength Diagram showing the results of seeding the prepared cells on a 3 ⁇ m pore PCL porous film and observing immunochemical staining for Nestin after 10 days in culture with a confocal laser microscope (Left figure). The figure on the right shows a Phalloidin fluorescence image taken at the same time.
  • FIG. 14a Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a 3 ⁇ m pore PCL porous film, and only the immunochemical staining for Nestin after 5 days in culture was observed with a confocal laser microscope.
  • FIG. 14a Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a 3 ⁇ m pore PCL porous film, and only the immunochemical staining for Nestin after 5 days in culture was observed with a confocal laser microscope.
  • FIG. 14b Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a 3 ⁇ m pore PCL porous film, and only the immunochemical staining for Nestin after 7 days in culture was observed with a confocal laser microscope.
  • FIG. 14b Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a 3 ⁇ m pore PCL porous film, and only the immunochemical staining for Nestin after 7 days in culture was observed with a confocal laser microscope.
  • FIG. 14c Mouse embryonic cerebral cortex tissue power Figure 1 shows the result of seeding the prepared cells on a 3 ⁇ m pore PCL porous film and observing only immunochemical staining for Nestin after 10 days in culture with a confocal laser microscope It is.
  • FIG. 15a Mouse embryonic cerebral cortex tissue strength The prepared cells were seeded on a PCL porous film with a pore size of 5 ⁇ m, and immunochemical staining for Nestin after 5 days in culture was observed with a confocal laser microscope. Yes (left). The right figure shows the fluorescence image of Phalloidin performed simultaneously.
  • FIG. 15b Mouse embryonic cerebral cortical tissue strength The prepared cells were seeded on a PCL porous film with a pore size of 8 ⁇ m, and immunochemical staining for Nestin after 5 days in culture was observed with a confocal laser microscope. Yes (left). The right figure shows the fluorescence image of Phalloidin performed simultaneously.
  • FIG. 15c PCL porous film with a pore size of 10 ⁇ m was prepared from mouse fetal cerebral cortical tissue strength. It is a figure which shows the result of having been seed
  • the present inventors cast a dilute mixed solution of a biodegradable polymer and an amphiphilic polymer newly synthesized by self-organization of the polymer onto a petri dish to obtain high humidity. So far, it has been found that a porous film having a regular porous structure can be produced by blowing air, and that the porous film can be used as a substrate for cell culture. Therefore, the present inventors have intensively studied for the purpose of finding a new use based on the unknown characteristics of the above-mentioned film and completing a more preferable culture substrate. Completed.
  • cell is primarily intended for animal cells, but may be plant cells.
  • Preferred cells are mammalian cells, more preferably human cells.
  • the present invention provides a culture substrate for growing stem cells without differentiation.
  • the culture substrate according to the present invention preferably comprises a thin film having a thickness in the range of 0.01 to L00 m.
  • the thin film may be laminated on a substrate (for example, plastic, glass, etc.) which may be a single layer or a plurality of thin films.
  • the thin film is made of coconut.
  • the above-mentioned coagulant is not particularly limited, but considering that it is used for culture, one having less toxicity is preferable.
  • the culture substrate according to the present embodiment is produced according to the method described in Patent Document 2, it is preferably a polymer compound (polymer) that is soluble in an organic solvent.
  • a polymer is formed in a desired manner using a printing method such as an ink jet method or a screen method in order to form a polymer thin film.
  • the shape and size can be paste, or the surface can be further refined using a photolithographic method. Shape it.
  • a support (substrate) is required, but the substrate is not particularly limited as long as it is dimensionally stable.
  • the substrate is not particularly limited as long as it is dimensionally stable.
  • plastic eg, polyethylene, polypropylene, polystyrene, etc.
  • metal plate eg, aluminum, zinc, copper, etc.
  • plastic film eg, cellulose diacetate, cellulose triacetate, etc.
  • Cellulose propionate cellulose butyrate, cellulose acetate butyrate, cellulose nitrate, polyethylene terephthalate, polyethylene, polystyrene, polypropylene, polycarbonate, polybulacetal, etc.
  • These may be a single component sheet such as a resin film or a metal plate, or may be a laminate of two or more materials.
  • polymers examples include polybutadiene, polyisoprene, styrene butadiene copolymer, conjugated gen-based high molecules such as acrylonitrile-butadiene-styrene copolymer; poly ⁇ -strength prolataton; polyurethane; Cellulose polymers such as nitrate cellose, acetyl cellulose, and cellophane; polyamide polymers such as polyamide 6, polyamide 66, polyamide 610, polyamide 612, polyamide 12, polyamide 46; polytetrafluoroethylene, Fluoropolymers such as polytrifluoroethylene and perfluoroethylene propylene copolymer; polystyrene, styrene ethylene propylene copolymer, styrene-ethylene-butylene copolymer, styrene-isoprene copolymer, chlorination Polyethylene Styrene polymers such as N
  • Polymers such as phenol resin, amino resin, urea resin, melamine resin, benzoguanamine resin; polymers such as polybutylene terephthalate, polyethylene terephthalate, polyethylene naphthalate E ester polymer, epoxy ⁇ ; poly (meth) acrylic acid esters, poly - 2-hydroxy-E (Meth) acrylic polymers such as tilatalylate, methacrylic acid ester and butyl acetate copolymer; norbornene-based resin; silicon resin; polymers of hydroxycarboxylic acids such as polylactic acid, polyhydroxybutyric acid, and polyglycolic acid These may be used alone or in combination.
  • the polymer constituting the culture substrate according to the present invention may be non-biodegradable or biodegradable!
  • in vitro amplification of stem cells in vitro is possible. It does not have to be biodegradable when the intended culture is performed.
  • non-biodegradable resin May be used.
  • Preferred biodegradable resins include polylactic acid, poly-force prolatatone), and poly (glycolic acid-lactic acid) copolymers
  • preferred non-biodegradable resins include polybutadiene, polyurethane, and poly ( (Meta) attalate.
  • the resin is composed of an amphiphilic polymer in the culture substrate according to this embodiment.
  • a preferred amphiphilic polymer is a polyethylene glycol z polypropylene glycol block copolymer; an acrylamide polymer as a main chain skeleton, a hydrophobic side chain as a dodecyl group, and a hydrophilic side chain as a ratato group or a carboxyl group.
  • amphipathic fats ion complexes of heroin dextran sulfate, nucleo acids (DNA and RNA) and long chain alkyl ammonium salts; gelatin, collagen, albumin Amphiphilic rosin based on water-soluble proteins such as polylactic acid, polyethylene glycol block copolymer, poly ⁇ -force prolatatone-polyethylene glycol block copolymer, polymalic acid-polymalic acid alkyl ester block copolymer Power that can be cited as amphiphilic rosin But are not limited to, et al. Are.
  • the culture substrate according to the present invention preferably has a plurality of pores.
  • the above-mentioned hole may be either a through-hole or a non-through-hole, as long as it has a porous structure at least on the surface portion.
  • each of the plurality of pores has a “continuous porous structure” in which the inside of the substrate communicates.
  • the average pore diameter of the pores is 0.1 to 20 ⁇ m.
  • the opening shape of the hole is not particularly limited, and may be any shape such as a circular shape, an elliptical shape, a square shape, a rectangular shape, or a hexagonal shape.
  • the term "hole diameter” intends the diameter of the largest inscribed circle with respect to the opening shape of the hole, for example, where the opening shape of the hole is substantially circular. Is intended to be the diameter of the circle, intended to be the minor axis of the ellipse if it is substantially elliptical, and intended to be the length of the side of the square if it is substantially square. In the case of a rectangular shape, the length of the short side of the rectangle is intended.
  • the stem width is 0.01 to 7 ⁇ m.
  • stem width is intended to be the width between holes.
  • the plurality of holes are regularly arranged, and more preferably, the plurality of holes are arranged in a Harkham-like manner.
  • the term “no-cam” (no-cam-like structure) is intended to mean a porous structure in which a plurality of pores having a substantially constant pore diameter are arranged in a regular honeycomb shape. .
  • a mold technology including nanoimprints (a hammer having a uniform pore size of about submicron to 100 microns) can be used as a method for producing a her cam-like structure.
  • a technique for obtaining a structure a method of drying a colloidal fine particle dispersion, and obtaining a Herkam-like porous film by using the accumulated colloidal crystals as a mold are known.
  • the shape of the voids collapses when the saddle is peeled off, so in principle it is difficult to accurately reflect the saddle structure.
  • time is required for integration. Such as the need to remove the mold after pouring the material.
  • the method for producing the culture substrate according to the present invention is as follows. It is not limited to this. [0066]
  • the subject using the culture substrate according to the present invention is not particularly limited as long as it is a stem cell, and may be any of a neural stem cell, a hematopoietic stem cell, a mesenchymal stem cell, a somatic stem cell, or an embryonic stem cell. .
  • hematopoietic stem cells hematopoietic malignant tumor, hematopoietic insufficiency, immunodeficiency, metabolic disease, solid tumor, etc.
  • Mesenchymal stem cells bone regeneration after fracture, muscle disease, ischemic defect, etc.
  • neural stem cells peripheral nerve injury (trauma), ischemia, nervous system malignant tumor, neurodegenerative disease, etc.), and basic research Stages of liver stem cells (liver failure), muscle stem cells (muscle disease), spleen stem cells (diabetes), skin stem cells (after burns, skin resection), retinal stem cells (retinal degenerative disease), and hair follicle stem cells (hair loss) (The main target disease is shown in parentheses).
  • neural stem cells include Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and cerebral infarction.
  • the treatment target sites are midbrain substantia nigra dopamine neurons, motor neurons, oligodendrocytes, neurons and Z or glia, respectively.
  • the culture substrate according to the present invention is Biodegradable materials are required to be applied to medical applications such as artificial organs in combination with cell engineering and cell culture technology.
  • the culture substrate according to the present invention cells can be cultured without serum, so that desired cells for use in autologous transplantation can be supplied safely. If the culture substrate according to the present invention is used, cells can be cultured without using growth factors, so that desired cells can be supplied at low cost.
  • an object of the present invention is to provide a culture substrate for proliferating stem cells without differentiating them.
  • the method for producing a thin film specifically described in the present specification, the thin film It does not depend on conditions such as the thickness of the resin, the composition of the oil, the number and depth of the holes, and the shape of the holes. Therefore, it should be noted that culture substrates produced using methods other than those described above are also within the scope of the present invention.
  • the present invention provides a culture substrate for differentiating stem cells.
  • the culture substrate according to the present invention preferably comprises a thin film having a film thickness in the range of 0.01 to LOO m.
  • the thin film may be laminated on a substrate (for example, plastic, glass, etc.) which may be a single layer or a plurality of thin films.
  • the thin film is made of coconut.
  • the above-mentioned coagulant is not particularly limited, but considering that it is used for culture, one having less toxicity is preferable.
  • the culture substrate according to the present embodiment is produced according to the method described in Patent Document 2, it is preferably a polymer compound (polymer) that is soluble in an organic solvent.
  • a polymer is formed in a desired manner using a printing method such as an ink jet method or a screen method in order to form a polymer thin film.
  • the shape and size can be paste, or the surface can be further shaped using a photolithographic method.
  • a support (substrate) is required, but the substrate is not particularly limited as long as it is dimensionally stable.
  • the substrate is not particularly limited as long as it is dimensionally stable.
  • plastic eg, polyethylene, polypropylene, polystyrene, etc.
  • metal plate eg, aluminum, zinc, copper, etc.
  • plastic film eg, cellulose diacetate, cellulose triacetate, propionic acid
  • These can be single component sheets such as resin films or metal plates
  • a laminate of two or more materials may be used.
  • polymers examples include polybutadiene, polyisoprene, styrene-butadiene copolymer, acrylonitrile-butadiene-styrene copolymer and other high molecular conjugation polymers; Cellulose polymers such as cellulose, acetyl cellulose, cellophane, etc .; Polyamide polymers such as polyamide 6, polyamide 66, polyamide 610, polyamide 612, polyamide 12, polyamide 46; polytetrafluoroethylene, polytrifluoro Fluoropolymers such as low ethylene, perfluoroethylene propylene copolymer; polystyrene, styrene ethylene propylene copolymer, styrene / ethylene / butylene copolymer, styrene / isoprene copolymer, chlorinated polyethylene— Styrene polymers such as acrylonitrile styrene copolymer, me
  • Polymers such as phenol resin, amino resin, urea resin, melamine resin, and benzoguanamine resin; polyesters such as polybutylene terephthalate, polyethylene terephthalate, and polyethylene naphthalate Terpolymers; epoxy resins; poly (meth) acrylic acid esters, poly-2-hydroxyethyl acrylate, methacrylic acid esters (meth) acrylic polymers such as butyl acetate copolymer; norbornene resin Silicone resin; examples include polymers of hydroxycarboxylic acids such as polylactic acid, polyhydroxybutyric acid, and polyglycolic acid, which are used alone. It can be used in combination.
  • the polymer constituting the culture substrate according to the present invention may be a non-biodegradable or a biodegradable resin, but it does not allow in vitro amplification of stem cells in vitro. It does not have to be biodegradable when performing the intended culture. In addition, when it is preferable to maintain the effect of the culture substrate for a long period of time in a living body, it is sufficient to use non-biodegradable resin. May be used.
  • Preferred biodegradable resins include polylactic acid, poly-force prolatatone), and poly (glycolic acid-lactic acid) copolymers, and preferred non-biodegradable resins include polybutadiene, polyurethane, and poly ( (Meta) attalate.
  • the resin includes an amphiphilic polymer.
  • a preferred amphiphilic polymer is a polyethylene glycol z polypropylene glycol block copolymer; an acrylamide polymer as a main chain skeleton, a hydrophobic side chain as a dodecyl group, and a hydrophilic side chain as a ratato group or a carboxyl group.
  • amphipathic fats ion complexes of heroin dextran sulfate, nucleo acids (DNA and RNA) and long chain alkyl ammonium salts; gelatin, collagen, albumin Amphiphilic rosin based on water-soluble proteins such as polylactic acid, polyethylene glycol block copolymer, poly ⁇ -force prolatatone-polyethylene glycol block copolymer, polymalic acid-polymalic acid alkyl ester block copolymer Power that can be cited as amphiphilic rosin But are not limited to, et al. Are.
  • the culture substrate according to the present invention preferably has a plurality of pores.
  • the above-mentioned hole may be either a through-hole or a non-through-hole, as long as it has a porous structure at least on the surface portion.
  • each of the plurality of pores has a “continuous porous structure” in which the inside of the substrate communicates.
  • the average pore diameter of the pores is 0.1 to 20 ⁇ m.
  • the opening shape of the hole is not particularly limited, and may be any of a circular shape, an elliptical shape, a square shape, a rectangular shape, a hexagonal shape, and the like. It may be a shape.
  • the term "hole diameter” is intended to mean the diameter of the largest inscribed circle with respect to the opening shape of the hole, for example, where the opening shape of the hole is substantially circular. Is intended to be the diameter of the circle, intended to be the minor axis of the ellipse if it is substantially elliptical, and intended to be the length of the side of the square if it is substantially square. In the case of a rectangular shape, the length of the short side of the rectangle is intended.
  • the stem width is preferably 0.01 to 7 ⁇ m.
  • stem width is intended to be the width between holes.
  • the plurality of holes are regularly arranged, and more preferably, the plurality of holes are arranged in a Harkham-like manner.
  • the term “no-cam” (no-cam-like structure) is intended to mean a porous structure in which a plurality of pores having a substantially constant pore diameter are arranged in a regular honeycomb shape. .
  • a mold technology including nanoimprints (a hammer having a uniform pore size of about submicron to 100 microns) can be used as a method for producing a her cam-like structure.
  • a technique for obtaining a structure a method of drying a colloidal fine particle dispersion, and obtaining a Herkam-like porous film by using the accumulated colloidal crystals as a mold are known.
  • the shape of the voids collapses when the saddle is peeled off, so in principle it is difficult to accurately reflect the saddle structure.
  • time is required for integration. Such as the need to remove the mold after pouring the material. Therefore, as a simple method for producing a honeycomb-like structure in which the porous structure is regularly arranged, the method described in Patent Document 2 is most preferred.
  • the method for producing the culture substrate according to the present invention is as follows. It is not limited to this.
  • the subject using the culture substrate according to the present invention is not particularly limited as long as it is a stem cell, and may be any of a neural stem cell, a hematopoietic stem cell, a mesenchymal stem cell, a somatic stem cell, or an embryonic stem cell. .
  • the culture substrate according to the present invention cells can be cultured without serum, and thus desired cells for use in autologous transplantation can be supplied safely. If the culture substrate according to the present invention is used, cells can be cultured without using a differentiation-inducing factor, so that desired cells can be supplied at low cost.
  • an object of the present invention is to provide a culture substrate for differentiating stem cells.
  • the method for producing a thin film and the thickness of the thin film specifically described in the present specification are provided. It does not depend on the conditions such as the fat composition, the number and depth of the holes, and the shape of the holes. Therefore, it should be noted that culture substrates produced using methods other than those described above also belong to the scope of the present invention.
  • a PCL flat membrane was prepared by dropping the above mixed solution onto an 18 mm square cover glass and using a spin coater (MIKAS A) at 1000 rpm for 30 seconds.
  • the produced self-organized porous film was cut out and adhered to an 18 mm square cover glass (MATSU NAMI).
  • PCL porous film and PCL flat membrane were washed by immersing in 1 propanol (Wako) for 5 minutes, sterilized by ethanol and UV irradiation in a cell culture container 35mmZnon-treated polyne ne culture dish (IWAKI), Immerse in Poly (L-Lysin) solution (50mgZl Poly (L-Lysine) (Sigma), 0.1M boric acid (Wako) (pH8.3)) for 1 hour, then wash with sterilized water 3 times, Incubation was performed at 37 ° C for 1 hour in a medium containing FBS (Fetal Bovine Serum) (Opti-MEM, 10% FBS), and then subjected to cell culture.
  • FBS Fetal Bovine Serum
  • Nerve cells were prepared from cerebral cortex tissue of embryonic day 14 ICR mice as follows. First, the mouse power on the 14th day of pregnancy was also removed after removing the fetus. Furthermore, the cerebral cortex was separated from the cerebral hemisphere and collected in the medium (Opti-MEM, Gibco), and the cells were dispersed using a Pasteur pipette. Subsequently, the number of cells was counted using a hemocytometer, and Viability measurement by trypan blue (Gibco) staining was performed.
  • Mouse fetal cerebral cortex tissue strength The prepared cell suspension was seeded on a culture substrate so as to have a cell density of 2.0 10 4 cells 7 cm 2 . Blood at 37 ° C and 5% CO on the first day
  • Nerve cells cultured for 5 days were washed with PBS, and 4% paraformaldehyde ZPBS was prepared and left at room temperature for 1 hour to fix the cells. After washing 3 times with PBS (10 minutes each), add Blocking solution (5% goat serum in PBS, 2.5% BSA, 0.2% Triton—X 1 00) for 1 hour at room temperature Cells were incubated. The blocking solution was removed, and the cells were incubated with a primary antibody (anti-Nestin antibody (1: 1000 in PBS)) for 1 hour at room temperature. After washing 3 times with PBS (10 min each), the cells were incubated with Piotin® anti-mouse IgG (1: 1000 in PBS) for 1 hour at room temperature.
  • Blocking solution 5% goat serum in PBS, 2.5% BSA, 0.2% Triton—X 1 00
  • BrdU was incorporated into the nuclei of the growing cells. Then use 10% formalin in the room. Cells were fixed for 2 hours at temperature. Wash three times with PBS (10 minutes each), incubate in 2M HC1 solution at 37 ° C for 60 minutes, then twice with 0.1M HBO buffer (for 5 minutes)
  • the cells were then incubated for 1 hour at room temperature with blocking solution (5% goat serum in PBS, 2.5% BSA, 0.2% Triton-X 100). After removing the blocking solution, the cells were incubated with primary antibody (anti-BrdU mouse IgG (1: 1000 in PBS) for 1 hour at room temperature.
  • blocking solution 5% goat serum in PBS, 2.5% BSA, 0.2% Triton-X 100.
  • primary antibody anti-BrdU mouse IgG (1: 1000 in PBS
  • the cells were Incubate with Piotin® anti-mouse IgG (PBS trowel 1: 1000) for 1 hour at room temperature, then wash with PBS, then incubate cells with Alexa488 labeled avidin (1: 2000 in PBS) for 30 minutes After washing 3 times with PBS (10 minutes each) and once with distilled water, the sample was placed on a glass slide and mounted with Mounting media (KPL).
  • Piotin® anti-mouse IgG PBS trowel 1: 1000
  • Alexa488 labeled avidin 1: 2000 in PBS
  • the sample was placed on a glass slide and mounted with Mounting media (KPL).
  • Embryonic day 14 mouse fetal cerebral cortex tissue strength In the prepared cells, there are many neural stem cells. The effects of adhesion between neural stem cells and flat membranes or porous films on cell morphological changes and on cell proliferation or differentiation were examined by morphological observation using SEM and immunochemical staining.
  • neural stem cells maintained a relatively undifferentiated state on a porous film with a pore size of 3 ⁇ m, and at the same time self-proliferated to form spheroid-like aggregates, and the bottom.
  • the young neurons in contact with the cells gradually became mature cells, and the nerve protrusions were radially extended.
  • PCL flat membranes, or on porous films with a pore size of 5 m or more (8 m, 10 m) neural stem cells were differentiated and showed an adhesion form according to the shape of the substrate. .
  • the culture substrate according to the present invention is used, the morphology of the cells can be freely controlled. In addition, if the culture substrate according to the present invention is used, cells can be cultured without serum. Thus, desired cells for use in autologous transplantation can be safely supplied. If the culture substrate according to the present invention is used, cells can be cultured without using growth factors and differentiation-inducing factors, so that desired cells can be supplied at low cost. If the culture substrate according to the present invention is used, stem cells that have been difficult to self-amplify can be proliferated without differentiation. Therefore, the present invention is very useful in various medical fields such as gene therapy, organ transplantation, bone marrow transplantation, cancer therapy, or regenerative medicine.

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Abstract

La présente invention concerne une construction où la morphologie des cellules peut être librement régulée. Dans un mode de réalisation, l’invention décrit une construction qui est une base de culture où la différenciation ou la prolifération des cellules souches peut être librement régulée et qui comprend un mince film ayant une épaisseur de film allant de 0,01 à 100 µm ou deux tels films ou plus disposés les uns sur les autres en couches. Dans la culture de base selon le mode de réalisation décrit ci-dessus, on préfère que ces minces films soient constitués d'une résine.
PCT/JP2006/303909 2005-03-02 2006-03-01 Matériau de base pour la régulation de la différenciation/prolifération de cellules WO2006093207A1 (fr)

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JPWO2007097121A1 (ja) * 2006-02-21 2009-07-09 Ｓｃｉｖａｘ株式会社 スフェロイドおよびスフェロイド群並びにこれらの製造方法
JP2010521985A (ja) * 2007-03-19 2010-07-01 ヴァシフ・ハシルジ パターン付スタック型バイオマテリアルおよび／または組織工学スキャフォルド
WO2011052281A1 (fr) * 2009-10-30 2011-05-05 国立大学法人 東京大学 Procédé pour construire un réseau de sphéroïdes de nerfs
WO2012137830A1 (fr) * 2011-04-05 2012-10-11 国立大学法人広島大学 Kit de culture de cellules animales, procédé de culture de cellules animales, procédé de culture sélective de cellules animales et procédé de différentiation cellulaire
JP2012527866A (ja) * 2008-05-27 2012-11-12 オーフス ウニベルシテット 哺乳類の幹細胞の成長および分化のための生体適合性材料
JP2014138605A (ja) * 2014-03-05 2014-07-31 Aarhus Universitet 哺乳類の幹細胞の成長および分化のための生体適合性材料
WO2014208778A1 (fr) * 2013-06-28 2014-12-31 国立大学法人東北大学 Film mince nanométrique modelé de support de cellule
JP2016214148A (ja) * 2015-05-20 2016-12-22 住友電気工業株式会社 細胞培養担体及びこれを備える細胞シート
EP3162891A4 (fr) * 2014-06-26 2018-01-03 Zeon Corporation Procédé pour promouvoir la différenciation de cellules en culture et promoteur de différenciation de cellules en culture
WO2018061846A1 (fr) 2016-09-27 2018-04-05 富士フイルム株式会社 Procédé de production de tissu cellulaire, et film poreux
WO2018066512A1 (fr) * 2016-10-07 2018-04-12 株式会社バイオ未来工房 Procédé de séparation de cellules souches, procédé d'induction de différentiation, et application de réceptacle de culture cellulaire
JP2019157017A (ja) * 2018-03-15 2019-09-19 株式会社リコー 中空構造体
WO2020032041A1 (fr) * 2018-08-06 2020-02-13 日産化学株式会社 Système de culture cellulaire et procédé de production de masse cellulaire le mettant en œuvre
JP2020167966A (ja) * 2019-04-04 2020-10-15 大日本印刷株式会社 細胞培養基材の製造方法、細胞培養基材、細胞付細胞培養基材、細胞培養容器、及び細胞付細胞培養容器

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JP5397934B2 (ja) * 2006-02-21 2014-01-22 Ｓｃｉｖａｘ株式会社 スフェロイド群およびその製造方法
JP2010521985A (ja) * 2007-03-19 2010-07-01 ヴァシフ・ハシルジ パターン付スタック型バイオマテリアルおよび／または組織工学スキャフォルド
US8304237B2 (en) 2007-06-20 2012-11-06 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Method and device for forming biologic cell aggregates
DE102007028423A1 (de) * 2007-06-20 2008-12-24 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren und Vorrichtung zur Bildung von Aggregaten biologischer Zellen
JP2012527866A (ja) * 2008-05-27 2012-11-12 オーフス ウニベルシテット 哺乳類の幹細胞の成長および分化のための生体適合性材料
WO2011052281A1 (fr) * 2009-10-30 2011-05-05 国立大学法人 東京大学 Procédé pour construire un réseau de sphéroïdes de nerfs
JPWO2011052281A1 (ja) * 2009-10-30 2013-03-14 国立大学法人 東京大学 神経スフェロイドネットワークの構築方法
JP6011879B2 (ja) * 2011-04-05 2016-10-19 国立大学法人広島大学 動物細胞培養キット、動物細胞の培養方法、動物細胞の選択培養方法及び細胞分化方法
WO2012137830A1 (fr) * 2011-04-05 2012-10-11 国立大学法人広島大学 Kit de culture de cellules animales, procédé de culture de cellules animales, procédé de culture sélective de cellules animales et procédé de différentiation cellulaire
US10196610B2 (en) 2011-04-05 2019-02-05 Hiroshima University Animal cell culture kit, method for culturing animal cells, method for selective culture of animal cells and cell differentiation method
WO2014208778A1 (fr) * 2013-06-28 2014-12-31 国立大学法人東北大学 Film mince nanométrique modelé de support de cellule
JP2014138605A (ja) * 2014-03-05 2014-07-31 Aarhus Universitet 哺乳類の幹細胞の成長および分化のための生体適合性材料
EP3162891A4 (fr) * 2014-06-26 2018-01-03 Zeon Corporation Procédé pour promouvoir la différenciation de cellules en culture et promoteur de différenciation de cellules en culture
JP2016214148A (ja) * 2015-05-20 2016-12-22 住友電気工業株式会社 細胞培養担体及びこれを備える細胞シート
WO2018061846A1 (fr) 2016-09-27 2018-04-05 富士フイルム株式会社 Procédé de production de tissu cellulaire, et film poreux
JPWO2018061846A1 (ja) * 2016-09-27 2018-12-27 富士フイルム株式会社 細胞組織の製造方法、及び多孔フィルム
US11633523B2 (en) 2016-09-27 2023-04-25 Fujifilm Corporation Method for producing cell tissue, and porous film
WO2018066512A1 (fr) * 2016-10-07 2018-04-12 株式会社バイオ未来工房 Procédé de séparation de cellules souches, procédé d'induction de différentiation, et application de réceptacle de culture cellulaire
JP2019157017A (ja) * 2018-03-15 2019-09-19 株式会社リコー 中空構造体
JP7056260B2 (ja) 2018-03-15 2022-04-19 株式会社リコー 中空構造体
WO2020032041A1 (fr) * 2018-08-06 2020-02-13 日産化学株式会社 Système de culture cellulaire et procédé de production de masse cellulaire le mettant en œuvre
JP2020167966A (ja) * 2019-04-04 2020-10-15 大日本印刷株式会社 細胞培養基材の製造方法、細胞培養基材、細胞付細胞培養基材、細胞培養容器、及び細胞付細胞培養容器

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