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WO2006093207A1 - Base material for regulating the differentiation/proliferation of cells - Google Patents

Base material for regulating the differentiation/proliferation of cells 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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WO
WIPO (PCT)
Prior art keywords
culture substrate
substrate according
stem cells
cells
culture
Prior art date
Application number
PCT/JP2006/303909
Other languages
French (fr)
Japanese (ja)
Inventor
Masaru Tanaka
Akinori Tsuruma
Sada-Aki Yamamoto
Masatsugu Shimomura
Original Assignee
National University Corporation Hokkaido University
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 National University Corporation Hokkaido University filed Critical National University Corporation Hokkaido University
Priority to JP2007505992A priority Critical patent/JP4956753B2/en
Publication of WO2006093207A1 publication Critical patent/WO2006093207A1/en

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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

A construct whereby the morphology of cells can be freely regulated. In an embodiment, a construct, which is a culture base whereby the differentiation or proliferation of stem cells can be freely controlled, comprising one thin film having a film thickness ranging from 0.01 to 100 μm or two or more such films layered together. In the culture base according to the above-described embodiment, it is preferred that these thin films are made of a resin.

Description

明 細 書  Specification
細胞の分化 Z増殖を制御するための基材  Cell differentiation Substrates for controlling Z proliferation
技術分野  Technical field
[0001] 本発明は、細胞の形態を自由に制御することができる構造体に関するものであり、 より詳細には、本発明は、幹細胞の分ィ匕または増殖を自由に操作することができる構 造体に関するものである。 背景技術  [0001] 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
[0002] 生物では、未分化細胞から幹細胞を経て、血液細胞、免疫細胞、神経細胞、皮膚 組織などの機能的な分化細胞が形成される。種々の系列において、その分化 (すな わち、成熟）の誘導または制御に関する研究は、細胞移植 (例えば、骨髄移植)また は組織再生もしくは組織修復という応用面で注目されている。特に、自己複製能（自 己増幅能)および多分化能 (個体を形成する全ての細胞種へ分化する能力）を有す る胚性幹細胞（embryonic stem cell:ES細胞）は、これらの能力を利用した再生 医療 (例えば、所望の細胞および Zまたは組織を必要に応じて作製して生体内へ移 植する医療）における応用が期待されている。  In organisms, functional differentiated cells such as blood cells, immune cells, nerve cells, and skin tissues are formed from undifferentiated cells via stem cells. In various series, research on the induction or control of its differentiation (ie, maturation) has attracted attention in applications such as cell transplantation (eg, bone marrow transplantation) or tissue regeneration or tissue repair. In particular, 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. Application in 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.
[0003] 幹細胞については、主に造血幹細胞の研究がなされてきた力 胚性幹細胞の確立 に続いて、現在までに各細胞系列（例えば、肝臓、筋肉、皮膚、神経など）の幹細胞 も同定されている。これらの幹細胞を遺伝子レベルで改変してその機能を必要に応 じて修飾する技術を開発することが、難治性疾患 (例えば、ガンおよび変性疾患など )の治療法の開発につながると考えられる。実際に、造血幹細胞はすでに骨髄移植 に応用されており、マウス胚性幹細胞は遺伝子ターゲティングにおいて使用されてい る。さら〖こ、最近単離されたヒト胚性幹細胞は移植を介する臓器形成への応用が期 待されており、正常組織幹細胞はガン治療もしくは再生医学、または組織幹細胞を 標的とする遺伝子治療への応用も期待されて!、る。  [0003] Regarding 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!
[0004] これまで全能性を有する胚性幹細胞が特に注目されて、研究が進められて!/、る。  [0004] So far, embryonic stem cells having totipotency have attracted particular attention and research has been advanced!
すなわち、胚性幹細胞のみが唯一の全能性幹細胞であると従来考えられており、体 性幹細胞 (すなわち、造血幹細胞、神経幹細胞などの組織幹細胞）は臓器限定的な 再生能のみを有すると考えられて 、た。 In other words, it is traditionally considered that only embryonic stem cells are the only totipotent stem cells, and somatic stem cells (ie, tissue stem cells such as hematopoietic stem cells and neural stem cells) are limited to organs. It was thought to have only regenerative capacity.
[0005] しかし、近年いくつかの組織幹細胞が環境に応じた幅広い分ィ匕能を有することが示 され、現在では、哺乳動物において、ほとんどの組織に固有の幹細胞が存在し、そ の増殖および分ィ匕によって機能性の成熟細胞が供給され、その結果として、各組織 の恒常性が維持されるということが知られている。また、 Bjornsonらは培養神経幹細 胞からインビボで成熟血球細胞を産生させたことを報告している (非特許文献 1を参 照のこと)。  [0005] However, in recent years, it has been shown that several tissue stem cells have a wide range of functions depending on the environment, and in mammals, there are stem cells unique to most tissues, and their proliferation and It is known that functional mature cells are supplied by sorting, and as a result, the homeostasis of each tissue is maintained. Also, Bjornson et al. Reported that mature blood cells were produced in vivo from cultured neural stem cells (see Non-Patent Document 1).
[0006] しかし、幹細胞を用いる研究の大きな課題は、 、ずれの幹細胞もその数が非常に 少ないことである。非特許文献 1に示された結果は再現されていないものの、この知 見が事実であれば、培養および継代が可能な神経幹細胞を使用すれば造血幹細胞 をインビトロで増幅させることが可能になる。  [0006] However, a major problem in research using stem cells is that the number of stem cells is very small. Although the results shown in Non-Patent Document 1 are not reproduced, if this finding is true, hematopoietic stem cells can be amplified in vitro by using neural stem cells that can be cultured and passaged .
[0007] このように幹細胞を遺伝子治療、臓器移植、骨髄移植、ガン治療、または再生医学 へ応用しょうとする試みが数多くなされている力 幹細胞を分ィ匕させずに自己増幅さ せる技術はほとんど開発されていない。従って、幹細胞を自由に操作する技術を開 発することは非常に望まれて 、る。 [0007] In this way, many attempts have been made to apply stem cells to gene therapy, organ transplantation, bone marrow transplantation, cancer treatment, or regenerative medicine. Most of the technologies to self-amplify stem cells without separating them. Not developed. Therefore, it is highly desirable to develop a technique for manipulating stem cells freely.
[0008] 細胞は、増殖因子またはサイト力インなどの液性因子だけでなぐ種々の高分子か らなる細胞外基質との接着によってその増殖および Zまたは分ィ匕が制御されている ことが知られている。この細胞と細胞外基質との相互作用において、細胞は、細胞外 基質分子の化学的な性質だけでなく、基質を構成する高分子が織り成す微細な形 状によってその増殖および Zまたは分ィ匕が制御されることもまた知られている。 [0008] It is known that the growth and Z or fractionation of cells is controlled by adhesion to an extracellular matrix composed of various macromolecules consisting only of growth factors or humoral factors such as cyto force-in. It has been. In this interaction between the cell and the extracellular matrix, the cell has not only the chemical nature of the extracellular matrix molecule, but also its growth and Z or fractionation due to the fine shape woven by the macromolecules that make up the matrix. It is also known to be controlled.
[0009] 組織を構築および Zまたは再生する際もまた、その組織の細胞および液性因子だ けでなく細胞の足場 (scaffold)を検討することが重要であり、組織工学の分野にお いては足場の開発が活発に行われている。このように、細胞外基質は細胞の特性を 制御する足場として細胞工学および糸且織工学の分野において注目されており、特に 特定の 3次元構造を有する人工基質の開発に向けて、多くの研究がなされている。 例えば、半導体技術において利用されているマイクロパターンなどが 3次元構造の製 造に利用されている。また、足場の材料として生体適合性および生分解性の高分子 が用いられており、材料表面のマイクロパターンが細胞の分化、増殖、形態に大きく 影響を及ぼして 、ることが報告されて 、る。 [0009] When constructing and regenerating or regenerating a tissue, it is important to consider not only the cells and humoral factors of the tissue but also the cell scaffold, and in the field of tissue engineering, Scaffolding is being actively developed. In this way, extracellular matrix is attracting attention in the field of cell engineering and yarn and weaving engineering as a scaffold for controlling the characteristics of cells. In particular, many studies have been conducted toward the development of artificial substrates having a specific three-dimensional structure. Has been made. For example, micropatterns used in semiconductor technology are used to manufacture three-dimensional structures. In addition, biocompatible and biodegradable polymers are used as scaffold materials, and the micro-pattern on the material surface greatly affects cell differentiation, proliferation, and morphology. It has been reported that it has an influence.
[0010] このように、足場が、人工物であってもその三次元構造に基づ 、て細胞の接着、増 殖および Zまたは分ィ匕を誘導し得ること、ならびに細胞を組込んだ足場を生体に移 植することによって組織を再構築し得ることが見出されている。神経細胞を標的とした 場合、微細加工技術によって作製された種々のマイクロパターン基板を用いることに よって、神経細胞の接着形態、神経突起の伸長を制御し、人工的な神経回路を構築 することが検討されており、神経再生への応用が期待される。  [0010] Thus, even if 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. When targeting neuronal cells, 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.
[0011] しかし、マイクロパターン技術は、非常に高度な技術が必要であり、大量生産が出 来ず、高コストであるといった多くの問題を抱えて 、る。  [0011] However, the micropattern technology requires a very high level of technology, has many problems such as high mass production and high cost.
[0012] 本発明者は、生分解性高分子と両親媒性ポリマーとを適当な割合で組み合わせる ことによって、経済的な調製が可能であり、自立性が有り、構造的にも安定な構造体 を作製し、該構造体を用いた細胞培養用基材を完成させている（例えば、特許文献 1 および 2を参照のこと)。  [0012] 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).
特許文献 1 :特開 2002— 347107公報（平成 14年 12月 4日公開）  Patent Document 1: Japanese Patent Laid-Open No. 2002-347107 (published on December 4, 2002)
特許文献 2：特開 2002— 335949公報（平成 14年 11月 26日公開）  Patent Document 2: JP 2002-335949 A (published on November 26, 2002)
非特許文献 l : Bjornson, C. R.ら、 Science 283 : 534— 537 (1999)。  Non-patent literature l: Bjornson, C. R. et al., Science 283: 534-537 (1999).
[0013] 上述したように、幹細胞を遺伝子治療、臓器移植、骨髄移植、ガン治療、または再 生医学へ応用しょうとする試みが数多くなされている。しかし、幹細胞を用いる研究の 大きな課題は、幹細胞の数が非常に少なぐ幹細胞特異的マーカーも発見されてい ないことである。そのため、幹細胞を純ィ匕することは困難であり、また増殖因子を添カロ せずに、幹細胞を分化させずに自己増殖させる技術も開発されていない。また、分 化誘導因子を添加せずに細胞の分ィヒを制御する技術も開発されていない。  [0013] 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. However, 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. In addition, a technique for controlling cell differentiation without adding a differentiation-inducing factor has not been developed.
[0014] 本発明は、上記の問題点に鑑みてなされたものであり、その目的は、細胞の形態を 自由に制御することができる構造体を提供するとともに、幹細胞の分化と増殖とを自 由に操作することができる構造体を提供することにある。  [0014] 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.
発明の開示  Disclosure of the invention
[0015] 本発明に係る培養基材は、幹細胞を分化させることなく増殖させることを特徴として いる。 [0016] 本発明に係る培養基材は、 0. 01〜： LOO mの範囲の膜厚を有している薄膜を備 えていることが好ましい。 [0015] The culture substrate according to the present invention is characterized in that stem cells are proliferated without differentiation. [0016] The culture substrate according to the present invention preferably includes a thin film having a film thickness ranging from 0.01 to LOO m.
[0017] 本発明に係る培養基材は、上記薄膜を複数積層して備えて ヽることが好ま ヽ。 [0017] It is preferable that the culture substrate according to the present invention comprises a plurality of the above thin films.
[0018] 本発明に係る培養基材は榭脂からなることが好ま 、。 [0018] The culture substrate according to the present invention is preferably made of sallow.
[0019] 本発明に係る培養基材において、上記榭脂は生分解性ポリマーを含むことが好ま しい。  [0019] In the culture substrate according to the present invention, it is preferable that the scab comprises a biodegradable polymer.
[0020] 本発明に係る培養基材において、上記榭脂は両親媒性ポリマーをさらに含むこと が好ましい。  [0020] In the culture substrate according to the present invention, it is preferable that the scab further contains an amphiphilic polymer.
[0021] 本発明に係る培養基材にお!ヽて、上記榭脂は生分解性ポリマーおよび両親媒性 ポリマーからなることが好まし!/、。  [0021] In the culture substrate according to the present invention, it is preferable that the above-mentioned coconut resin comprises a biodegradable polymer and an amphiphilic polymer!
[0022] 本発明に係る培養基材にお!ヽて、上記生分解性ポリマーは、ポリ乳酸、ポリ（ ε— 力プロラタトン)、およびポリ（グリコール酸-乳酸)共重合体からなる群より選択される ことが好ましい。 [0022] In the culture substrate according to the present invention, the biodegradable polymer is selected from the group consisting of polylactic acid, poly (ε-force prolatatone), and poly (glycolic acid-lactic acid) copolymer. It is preferable that
[0023] 本発明に係る培養基材にお!、て、上記両親媒性ポリマーは、疎水性側鎖としてド デシル基を有し親水性側鎖としてラタトース基またはカルボキシル基を有して 、る、ァ クリルアミドポリマーを主鎖骨格とする両親媒性榭脂；ポリエチレングリコール系共重 合体；および、ァ-オン性高分子と長鎖アルキルアンモ-ゥム塩とのポリイオンコンプ レックスポリイオンコンプレックス力もなる群より選択されることが好ましい。  [0023] In the culture substrate according to the present invention, 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.
[0024] 本発明に係る培養基材は複数の孔を有して ヽることが好ま 、。 [0024] The culture substrate according to the present invention preferably has a plurality of pores.
[0025] 本発明に係る培養基材において、平均孔径が 0. 1〜20 μ mであることが好ましい [0025] In the culture substrate according to the present invention, the average pore diameter is preferably 0.1 to 20 µm.
[0026] 本発明に係る培養基材において、孔径の変動係数が 30%以下であることが好まし い。 [0026] In the culture substrate according to the present invention, the coefficient of variation in pore diameter is preferably 30% or less.
[0027] 本発明に係る培養基材において、孔と孔との間の幅が 0. 01〜7 μ mであることが 好ましい。  [0027] In the culture substrate according to the present invention, the width between the pores is preferably 0.01 to 7 µm.
[0028] 本発明に係る培養基材にお!、て、上記複数の孔はハ-カム様に配列されて ヽるこ とが好ましい。  [0028] In the culture substrate according to the present invention, it is preferable that the plurality of holes are arranged in a Harkham-like manner.
[0029] 本発明に係る培養基材にお!、て、各孔は貫通して!/ヽることが好ま ヽ。 [0030] 本発明に係る培養基材にお!、て、各孔は連通して ヽることが好まし!/ヽ。 [0029] It is preferable that each hole penetrates the culture substrate according to the present invention. [0030] In the culture substrate according to the present invention, it is preferable that the holes communicate with each other!
[0031] 本発明に係る培養基材にお!ヽて、上記幹細胞は、神経幹細胞、造血幹細胞、間葉 系幹細胞、体性幹細胞および胚性幹細胞力 なる群より選択されることが好ま U、。  [0031] In the culture substrate according to the present invention, 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, .
[0032] 本発明に係る培養基材は、幹細胞を分化させることを特徴として ヽる。 [0032] The culture substrate according to the present invention is characterized by differentiating stem cells.
[0033] 本発明に係る培養基材は、 0. 01〜： LOO mの範囲の膜厚を有している薄膜を備 えていることが好ましい。 [0033] 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.
[0034] 本発明に係る培養基材は、上記薄膜を複数積層して備えて ヽることが好ま ヽ。 [0034] It is preferable that the culture substrate according to the present invention comprises a plurality of the above thin films.
[0035] 本発明に係る培養基材は榭脂からなることが好ま 、。 [0035] It is preferable that the culture substrate according to the present invention comprises sallow.
[0036] 本発明に係る培養基材にお!ヽて、上記榭脂は生分解性ポリマーを含むことが好ま しい。  [0036] In the culture substrate according to the present invention, it is preferable that the coconut resin contains a biodegradable polymer.
[0037] 本発明に係る培養基材において、上記榭脂は両親媒性ポリマーをさらに含むこと が好ましい。  [0037] In the culture substrate according to the present invention, it is preferable that the scab further contains an amphiphilic polymer.
[0038] 本発明に係る培養基材にお!ヽて、上記榭脂は生分解性ポリマーおよび両親媒性 ポリマーからなることが好まし!/、。  [0038] In the culture substrate according to the present invention, it is preferable that the above-mentioned coconut resin comprises a biodegradable polymer and an amphiphilic polymer!
[0039] 本発明に係る培養基材にお!ヽて、上記生分解性ポリマーは、ポリ乳酸、ポリ（ ε— 力プロラタトン)、およびポリ（グリコール酸-乳酸)共重合体からなる群より選択される ことが好ましい。 [0039] In the culture substrate according to the present invention, the biodegradable polymer is selected from the group consisting of polylactic acid, poly (ε-force prolatatone), and poly (glycolic acid-lactic acid) copolymer. It is preferable that
[0040] 本発明に係る培養基材にお!、て、上記両親媒性ポリマーは、疎水性側鎖としてド デシル基を有し親水性側鎖としてラタトース基またはカルボキシル基を有して 、る、ァ クリルアミドポリマーを主鎖骨格とする両親媒性榭脂；ポリエチレングリコール系共重 合体；および、ァ-オン性高分子と長鎖アルキルアンモ-ゥム塩とのポリイオンコンプ レックス力もなる群より選択されることが好ま 、。  [0040] In the culture substrate according to the present invention, 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. From the group consisting of 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.
[0041] 本発明に係る培養基材は複数の孔を有して ヽることが好ま 、。 [0041] The culture substrate according to the present invention preferably has a plurality of pores.
[0042] 本発明に係る培養基材にお!、て、平均孔径が 0. 1〜20 μ mであることが好まし ヽ [0042] The culture substrate according to the present invention preferably has an average pore diameter of 0.1 to 20 µm.
[0043] 本発明に係る培養基材において、孔径の変動係数が 30%以下であることが好まし い。 [0043] In the culture substrate according to the present invention, the pore diameter variation coefficient is preferably 30% or less.
[0044] 本発明に係る培養基材において、孔と孔との間の幅が 0. 01〜7 μ mであることが 好ましい。 [0044] In the culture substrate according to the present invention, the width between the pores is 0.01 to 7 µm. preferable.
[0045] 本発明に係る培養基材にお!、て、上記複数の孔はハ-カム様に配列されて ヽるこ とが好ましい。  [0045] In the culture substrate according to the present invention, it is preferable that the plurality of holes are arranged in a Herkam-like manner.
[0046] 本発明に係る培養基材にお!、て、各孔は貫通して!/ヽることが好ま ヽ。  [0046] It is preferable that each of the holes penetrates the culture substrate according to the present invention.
[0047] 本発明に係る培養基材にお!、て、各孔は連通して ヽることが好ま ヽ。 [0047] In the culture substrate according to the present invention, it is preferable that the holes communicate with each other.
[0048] 本発明に係る培養基材にお!ヽて、上記幹細胞は、神経幹細胞、造血幹細胞、間葉 系幹細胞、体性幹細胞および胚性幹細胞力 なる群より選択されることが好ま U、。 [0048] In the culture substrate according to the present invention, 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. .
[0049] 本発明のさらに他の目的、特徴、および優れた点は、以下に示す記載によって十 分わ力るであろう。また、本発明の利益は、添付図面を参照した次の説明で明白にな るであろう。 [0049] Still other objects, features, and advantages of the present invention will be sufficiently enhanced by the following description. The benefits of the present invention will become apparent from the following description with reference to the accompanying drawings.
図面の簡単な説明  Brief Description of Drawings
[0050] [図 1]本発明の一実施形態に係る多孔フィルムの形状、ならびに該フィルムにおいて 測定した孔径、幹径および空孔率を示す図である。  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.
[図 2a]マウス胎仔大脳皮質組織力ゝら調製した細胞を PCL平膜上に播種し、 4時間後 に Nestinに対する免疫化学染色を行った結果を示す図である (左図）。右図は、同 時に行った Phalloidinの蛍光像を示す。  [Fig. 2a] 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.
[図 2b]マウス胎仔大脳皮質組織力 調製した細胞を PCL平膜上に播種し、 4時間後 に BudUに対する免疫化学染色を行った結果を示す図である (左図）。右図は、同時 に行った Phalloidinの蛍光像を示す。  [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.
[図 3a]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの多孔フィルム上に 播種し、 4時間後に Nestinに対する免疫化学染色を行った結果を示す図である (左 図）。右図は、同時に行った Phalloidinの蛍光像を示す。  [FIG. 3a] Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a porous film having a pore diameter of 3 μm, and immunochemical staining for Nestin was performed 4 hours later (left figure). The right figure shows the fluorescence image of Phalloidin performed simultaneously.
[図 3b]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの多孔フィルム上に 播種し、 4時間後に BudUに対する免疫化学染色を行った結果を示す図である（左 図）。右図は、同時に行った Phalloidinの蛍光像を示す。  [Fig. 3b] Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a porous film having a pore size of 3 μm, and immunochemical staining for BudU was performed 4 hours later (left). The right figure shows the fluorescence image of Phalloidin performed simultaneously.
[図 4a]マウス胎仔大脳皮質組織力ゝら調製した細胞を PCL平膜上に播種し、培養 5日 後の細胞の形態を走査型電子顕微鏡で観察した結果を示す図である。  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.
[図 4b]マウス胎仔大脳皮質組織力 調製した細胞を孔径 2〜3 μ mの PCL多孔フィ ルム上に播種し、培養 5日後の細胞の形態を走査型電子顕微鏡で観察した結果を 示す図である。 [Fig. 4b] Mouse fetal cerebral cortex tissue strength The prepared cells were placed in PCL porous membranes with a pore size of 2-3 μm. 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.
圆 5a]マウス胎仔大脳皮質組織力ゝら調製した細胞を PCL平膜上に播種し、培養 5日 後の β -tubulin ΠΙによる免疫化学染色を共焦点レーザー顕微鏡にて観察した結 果を示す図である。 [5a] Mouse embryonic cerebral cortex tissue strength seeded cells were seeded on PCL flat membrane and immunochemical staining with β-tubulin sputum after 5 days in culture was observed with a confocal laser microscope. It is.
圆 5b]マウス胎仔大脳皮質組織力ゝら調製した細胞を PCL平膜上に播種し、培養 5日 後の細胞の形態を走査型電子顕微鏡にて観察した結果を示す図である。 [5b] Mouse fetal cerebral cortex tissue strength cells were seeded on a PCL flat membrane, and the cell morphology after 5 days in culture was observed with a scanning electron microscope.
[図 5c]bにて示した細胞の形態の模式図である。 FIG. 5c is a schematic diagram of the cell morphology shown in b.
[図 6a]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの PCL多孔フィルム 上に播種し、培養 5日後の j8 -tubulin IIIによる免疫化学染色を共焦点レーザー 顕微鏡にて観察した結果を示す図である。  [Fig. 6a] Mouse embryonic cerebral cortex tissue strength The prepared cells were seeded on a 3 μm pore PCL porous film, and immunochemical staining with j8-tubulin III was observed with a confocal laser microscope after 5 days in culture. FIG.
[図 6b]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの PCL多孔フィルム 上に播種し、培養 5日後の細胞の形態を走査型電子顕微鏡にて観察した結果を示 す図である。  [Fig. 6b] Mouse embryonic cerebral cortex tissue strength The prepared cells were seeded on a PCL porous film with a pore size of 3 μm, and the morphology of the cells after 5 days in culture was observed with a scanning electron microscope. .
[図 6c]bにて示した細胞の形態の模式図である。  FIG. 6c is a schematic diagram of the cell morphology shown in b.
圆 7a]マウス胎仔大脳皮質組織力も調製した細胞を孔径 5 μ mの PCL多孔フィルム 上に播種し、培養 5日後の j8 -tubulin IIIによる免疫化学染色を共焦点レーザー 顕微鏡にて観察した結果を示す図である。 [7a] Mouse fetal cerebral cortical tissue strength prepared cells were seeded on PCL porous film with a pore size of 5 μm, and immunochemical staining with j8-tubulin III after 5 days in culture is shown by confocal laser microscopy FIG.
圆 7b]マウス胎仔大脳皮質組織力も調製した細胞を孔径 5 μ mの PCL多孔フィルム 上に播種し、培養 5日後の細胞の形態を走査型電子顕微鏡にて観察した結果を示 す図である。 [7b] This is a figure showing the results of seeding cells prepared with mouse fetal cerebral cortical tissue on a PCL porous film with a pore size of 5 μm and observing the morphology of the cells after 5 days of culture with a scanning electron microscope.
[図 7c]bにて示した細胞の形態の模式図である。  FIG. 7c is a schematic diagram of the cell morphology shown in b.
[図 8a]マウス胎仔大脳皮質組織力 調製した細胞を孔径 8 μ mの PCL多孔フィルム 上に播種し、培養 5日後の j8 -tubulin IIIによる免疫化学染色を共焦点レーザー 顕微鏡にて観察した結果を示す図である。  [Fig. 8a] Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a PCL porous film with a pore size of 8 μm, and immunochemical staining with j8-tubulin III was observed with a confocal laser microscope after 5 days in culture. FIG.
[図 8b]マウス胎仔大脳皮質組織力 調製した細胞を孔径 8 μ mの PCL多孔フィルム 上に播種し、培養 5日後の細胞の形態を走査型電子顕微鏡にて観察した結果を示 す図である。 [図 8c]bにて示した細胞の形態の模式図である。 [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 μm 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.
[図 9a]マウス胎仔大脳皮質組織力も調製した細胞を孔径 10 μ mの PCL多孔フィルム 上に播種し、培養 5日後の j8 -tubulin IIIに対する免疫化学染色を共焦点レーザ 一顕微鏡にて観察した結果を示す図である。  [Fig. 9a] Cells seeded with mouse embryonic cerebral cortical tissue force were seeded on PCL porous film with a pore size of 10 μm, and immunochemical staining for j8-tubulin III after 5 days in culture was observed with a confocal laser microscope. FIG.
圆 9b]マウス胎仔大脳皮質組織力も調製した細胞を孔径 10 μ mの PCL多孔フィル ム上に播種し、培養 5日後の細胞の形態を走査型電子顕微鏡にて観察した結果を 示す図である。 [9b] This is a figure showing the results of observing the morphology of cells after 5 days in culture with a scanning electron microscope after seeding cells prepared with mouse embryonic cerebral cortical tissue strength on a PCL porous film with a pore size of 10 μm.
[図 9c]bにて示した細胞の形態の模式図である。  FIG. 9c is a schematic diagram of the cell morphology shown in b.
圆 10a]マウス胎仔大脳皮質組織力ゝら調製した細胞を PCL平膜上に播種し、培養 3 日後の Nestinに対する免疫化学染色を共焦点レーザー顕微鏡にて観察した結果を 示す図である。 [10a] Mouse fetal cerebral cortical tissue strength seeded cells were seeded on PCL flat membranes, and immunochemical staining for Nestin after 3 days in culture was observed with a confocal laser microscope.
圆 10b]マウス胎仔大脳皮質組織力ゝら調製した細胞を PCL平膜上に播種し、培養 7 日後の Nestinに対する免疫化学染色を共焦点レーザー顕微鏡にて観察した結果を 示す図である。 [10b] Mouse fetal cerebral cortex tissue strength cells were seeded on a PCL flat membrane, and the results of observation of immunochemical staining for Nestin after 7 days in culture with a confocal laser microscope are shown.
圆 11a]マウス胎仔大脳皮質組織力ゝら調製した細胞を PCL平膜上に播種し、培養 3 日後の BrdUに対する免疫化学染色を共焦点レーザー顕微鏡にて観察した結果を 示す図である。 [11a] Mouse fetal cerebral cortex tissue strength cells were seeded on PCL flat membrane, and immunochemical staining for BrdU after 3 days in culture was observed with a confocal laser microscope.
圆 lib]マウス胎仔大脳皮質組織力ゝら調製した細胞を PCL平膜上に播種し、培養 7 日後の BrdUに対する免疫化学染色を共焦点レーザー顕微鏡にて観察した結果を 示す図である。 [Lib] The cells prepared from mouse fetal cerebral cortical tissue force were seeded on PCL flat membranes, and the results of observation of immunochemical staining for BrdU after 7 days in culture with a confocal laser microscope are shown.
[図 12a]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの PCL多孔フィルム 上に播種し、培養 3日後の BrdUに対する免疫化学染色を共焦点レーザー顕微鏡に て観察した結果を示す図である。  [Fig. 12a] Mouse embryonic cerebral cortex tissue strength. The prepared cells were seeded on a PCL porous film with a pore size of 3 μm, and immunochemical staining for BrdU after 3 days in culture was observed with a confocal laser microscope. is there.
[図 12b]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの PCL多孔フィルム 上に播種し、培養 7日後の BrdUに対する免疫化学染色を共焦点レーザー顕微鏡に て観察した結果を示す図である。  [Fig. 12b] Mouse embryonic cerebral cortex tissue strength. The prepared cells were seeded on a PCL porous film with a pore size of 3 μm, and immunochemical staining for BrdU after 7 days in culture was observed with a confocal laser microscope. is there.
[図 13a]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの PCL多孔フィルム 上に播種し、培養 5日後の Nestinに対する免疫化学染色を共焦点レーザー顕微鏡 にて観察した結果を示す図である（左図）。右図は、同時に行った Phalloidinの蛍光 像を示す。 [Fig. 13a] Mouse embryonic cerebral cortex tissue strength Prepared cells were seeded on a 3 μm 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.
[図 13b]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの PCL多孔フィルム 上に播種し、培養 7日後の Nestinに対する免疫化学染色を共焦点レーザー顕微鏡 にて観察した結果を示す図である（左図）。右図は、同時に行った Phalloidinの蛍光 像を示す。  [Fig. 13b] Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a PCL porous film with a pore size of 3 μm, 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.
[図 13c]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの PCL多孔フィルム 上に播種し、培養 10日後の Nestinに対する免疫化学染色を共焦点レーザー顕微 鏡にて観察した結果を示す図である（左図）。右図は、同時に行った Phalloidinの蛍 光像を示す。  [Fig. 13c] Mouse embryonic cerebral cortical tissue strength Diagram showing the results of seeding the prepared cells on a 3 μm 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.
[図 14a]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの PCL多孔フィルム 上に播種し、培養 5日後の Nestinに対する免疫化学染色のみを共焦点レーザー顕 微鏡にて観察した結果を示す図である。  [Fig. 14a] Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a 3 μm pore PCL porous film, and only the immunochemical staining for Nestin after 5 days in culture was observed with a confocal laser microscope. FIG.
[図 14b]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの PCL多孔フィルム 上に播種し、培養 7日後の Nestinに対する免疫化学染色のみを共焦点レーザー顕 微鏡にて観察した結果を示す図である。  [Fig. 14b] Mouse fetal cerebral cortex tissue strength The prepared cells were seeded on a 3 μm pore PCL porous film, and only the immunochemical staining for Nestin after 7 days in culture was observed with a confocal laser microscope. FIG.
[図 14c]マウス胎仔大脳皮質組織力 調製した細胞を孔径 3 μ mの PCL多孔フィルム 上に播種し、培養 10日後の Nestinに対する免疫化学染色のみを共焦点レーザー 顕微鏡にて観察した結果を示す図である。  [Fig. 14c] Mouse embryonic cerebral cortex tissue power Figure 1 shows the result of seeding the prepared cells on a 3 μm pore PCL porous film and observing only immunochemical staining for Nestin after 10 days in culture with a confocal laser microscope It is.
[図 15a]マウス胎仔大脳皮質組織力 調製した細胞を孔径 5 μ mの PCL多孔フィルム 上に播種し、培養 5日後の Nestinに対する免疫化学染色を共焦点レーザー顕微鏡 にて観察した結果を示す図である（左図）。右図は、同時に行った Phalloidinの蛍光 像を示す。  [FIG. 15a] Mouse embryonic cerebral cortex tissue strength The prepared cells were seeded on a PCL porous film with a pore size of 5 μm, 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.
[図 15b]マウス胎仔大脳皮質組織力 調製した細胞を孔径 8 μ mの PCL多孔フィルム 上に播種し、培養 5日後の Nestinに対する免疫化学染色を共焦点レーザー顕微鏡 にて観察した結果を示す図である（左図）。右図は、同時に行った Phalloidinの蛍光 像を示す。  [Fig. 15b] Mouse embryonic cerebral cortical tissue strength The prepared cells were seeded on a PCL porous film with a pore size of 8 μm, 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.
[図 15c]マウス胎仔大脳皮質組織力も調製した細胞を孔径 10 μ mの PCL多孔フィル ム上に播種し、培養 5日後の Nestinに対する免疫化学染色を共焦点レーザー顕微 鏡にて観察した結果を示す図である（左図）。右図は、同時に行った Phalloidinの蛍 光像を示す。 [Figure 15c] PCL porous film with a pore size of 10 μm was prepared from mouse fetal cerebral cortical tissue strength. It is a figure which shows the result of having been seed | inoculated on the mouse | mouth and observing the immunochemical staining with respect to Nestin 5 days after culture | cultivation with the confocal laser microscope (left figure). The figure on the right shows a Phalloidin fluorescence image taken at the same time.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0051] 上述したように、本発明者らは、生分解性高分子と高分子の自己組織化によって新 規合成した両親媒性高分子との希薄混合溶液をシャーレ上にキャストして高湿度の 空気を吹き付けることにより規則的な多孔構造を有する多孔性フィルムを作製するこ とができること、および該多孔性フィルムが細胞培養用基材として利用可能であること を、これまでに見出している。そこで、本発明者らは、上記フィルムが備えている未知 の特性に基づく新規用途を見出すこと、およびより好ましい培養基材を完成させるこ とを目的として鋭意検討を行い、その結果、本発明を完成させた。  [0051] As described above, 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.
[0052] なお、本明細書中で使用される場合、用語「細胞」は主に動物細胞が意図されるが 、植物細胞であってもよい。好ましい細胞は、哺乳動物細胞であり、より好ましくは、ヒ ト細胞である。  [0052] As used herein, the term "cell" is primarily intended for animal cells, but may be plant cells. Preferred cells are mammalian cells, more preferably human cells.
[0053] (1)幹細胞増殖用培養基材  [0053] (1) Culture substrate for stem cell proliferation
本発明は、幹細胞を分化させることなく増殖させるための培養基材を提供する。一 実施形態において、本発明に係る培養基材は、好ましくは 0. 01〜： L00 mの範囲 の膜厚を有している薄膜を備えている。本実施形態に係る培養基材において、上記 薄膜は 1層であっても複数積層されていてもよぐ基板 (例えば、プラスチック、ガラス など)上に該薄膜が積層されていてもよい。  The present invention provides a culture substrate for growing stem cells without differentiation. In one embodiment, 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. In the culture substrate according to the present embodiment, 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.
[0054] 本実施形態に係る培養基材にお!/、て、上記薄膜は榭脂からなることが好ま 、。本 実施形態に係る培養基材において、上記榭脂としては、特に限定されないが、培養 用途であることを考慮すると毒性の少ないものが好ましい。また、本実施形態に係る 培養基材を、特許文献 2に記載されるような方法に従って作製する場合は、有機溶 媒に溶解する高分子化合物 (ポリマー)であることが好ましい。本実施形態に係る培 養基材がポリマーカゝらなる薄膜を備えている場合、高分子薄膜を形成するために、ィ ンクジェット方式またはスクリーン方式のような印刷法を用いて高分子を所望の形状 およびサイズにペーストもよいし、フォトリソグラフィ一法などを用いてさらに表面を整 形してちょい。 [0054] In the culture substrate according to this embodiment, it is preferable that the thin film is made of coconut. In the culture substrate according to the present embodiment, the above-mentioned coagulant is not particularly limited, but considering that it is used for culture, one having less toxicity is preferable. Further, when 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. In the case where the culture substrate according to the present embodiment includes a thin film made of a polymer cover, 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.
[0055] なお、このような手法を用いてポリマー力もなる薄膜を形成する場合は、支持体 (基 板）が必要であるが、基板としては、寸度的に安定なものであれば特に限定されず、 例えば、紙、プラスチック（例えば、ポリエチレン、ポリプロピレン、ポリスチレン等）がラ ミネートされた紙、金属板 (例えば、アルミニウム、亜鉛、銅等）、プラスチックフィルム（ 例えば、二酢酸セルロース、三酢酸セルロース、プロピオン酸セルロース、酪酸セル ロース、酢酸酪酸セルロース、硝酸セルロース、ポリエチレンテレフタレート、ポリェチ レン、ポリスチレン、ポリプロピレン、ポリカーボネート、ポリビュルァセタール等）等が 挙げられる。これらは、榭脂フィルムまたは金属板などの単一成分のシートであっても 、 2以上の材料の積層体であってもよい。  [0055] When a thin film having a polymer force is formed using such a method, a support (substrate) is required, but the substrate is not particularly limited as long as it is dimensionally stable. For example, paper, paper laminated with 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.
[0056] このようなポリマーとしては、ポリブタジエン、ポリイソプレン、スチレン ブタジエン 共重合体、アクリロニトリル—ブタジエン—スチレン共重合体などの共役ジェン系高 分子；ポリ ε一力プロラタトン；ポリウレタン；酢酸セルロース、セルロイド、硝酸セル口 ース、ァセチルセルロース、セロファンなどのセルロース系高分子；ポリアミド 6、ポリア ミド 66、ポリアミド 610、ポリアミド 612、ポリアミド 12、ポリアミド 46などのポリアミド系高 分子；ポリテトラフルォロエチレン、ポリトリフルォロエチレン、パーフルォロエチレン プロピレン共重合体などのフッ素系高分子；ポリスチレン、スチレン エチレン プロ ピレン共重合体、スチレン一エチレン一ブチレン共重合体、スチレン一イソプレン共 重合体、塩素化ポリエチレン—アクリロニトリル スチレン共重合体、メタクリル酸エス テル スチレン共重合体、スチレン—アクリロニトリル共重合体、スチレン 無水マレ イン酸共重合体、アクリル酸エステル—アクリロニトリル スチレン共重合体などのス チレン系高分子；ポリエチレン、塩素化ポリエチレン、エチレン aーォレフイン共重 合体、エチレン 酢酸ビュル共重合体、エチレン一塩化ビュル共重合体、エチレン 酢酸ビュル共重合体、ポリプロピレン、ォレフィン ビュルアルコール共重合体、 ポリメチルペンテンなどのォレフィン系高分子;フエノール榭脂、アミノ榭脂、尿素榭 脂、メラミン榭脂、ベンゾグアナミン榭脂などのホルムアルデヒド系高分子;ポリブチレ ンテレフタレート、ポリエチレンテレフタレート、ポリエチレンナフタレートなどのポリェ ステル系高分子;エポキシ榭脂；ポリ（メタ)アクリル酸エステル、ポリ— 2—ヒドロキシェ チルアタリレート、メタクリル酸エステル 酢酸ビュル共重合体などの（メタ）アクリル系 高分子;ノルボルネン系榭脂；シリコン榭脂；ポリ乳酸、ポリヒドロキシ酪酸、ポリグリコ ール酸などのヒドロキシカルボン酸の重合体などが挙げられ、これらは単独で使用さ れても組み合わせて使用されてもょ 、。 [0056] Examples of such polymers include polybutadiene, polyisoprene, styrene butadiene copolymer, conjugated gen-based high molecules such as acrylonitrile-butadiene-styrene copolymer; poly ε-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-acrylonitrile styrene copolymer, methacrylate ester styrene copolymer, styrene-acrylonitrile copolymer, styrene-maleic anhydride copolymer, acrylic ester-acrylonitrile styrene copolymer; Olefins such as polyethylene, chlorinated polyethylene, ethylene a-olefin copolymer, ethylene acetate butyl copolymer, ethylene monochloride butyl copolymer, ethylene acetate butyl copolymer, polypropylene, olefin fin butyl alcohol copolymer, polymethylpentene, etc. Polymers: Formaldehyde 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 榭脂; 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.
[0057] なお、本発明に係る培養基材を構成するポリマーは、非生分解性榭脂であっても 生分解性榭脂であってもよ!、が、生体外で幹細胞のインビトロ増幅を目的とした培養 を行う場合は、生分解性である必要はない。また、培養基材の効果を生体内で長期 間持続させることが好ましい場合は、非生分解性榭脂を用いればよぐ生体で長期 間持続させることが好ましくない場合は、生分解性榭脂を用いればよい。好ましい生 分解性榭脂としては、ポリ乳酸、ポリ —力プロラタトン）、およびポリ（グリコール酸 —乳酸)共重合体が挙げられ、好ましい非生分解性榭脂としては、ポリブタジエン、 ポリウレタン、およびポリ（メタ）アタリレートが挙げられる。  [0057] It should be noted that the polymer constituting the culture substrate according to the present invention may be non-biodegradable or biodegradable! However, in vitro amplification of stem cells in vitro is possible. It does not have to be biodegradable when the intended culture is performed. 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.
[0058] 本実施形態に係る培養基材にお!/ヽて、上記樹脂が両親媒性のポリマーを含んで 構成されることが好ましい。本実施形態において、好ましい両親媒性ポリマーとして は、ポリエチレングリコール zポリプロピレングリコールブロック共重合体；アクリルアミ ドポリマーを主鎖骨格とし疎水性側鎖としてドデシル基と親水性側鎖としてラタトース 基またはカルボキシル基を併せ持つ両親媒性榭脂；へノ リンゃデキストラン硫酸、核 酸（DNAや RNA)などのァ-オン性高分子と長鎖アルキルアンモ-ゥム塩とのィォ ンコンプレックス；ゼラチン、コラーゲン、アルブミンなどの水溶性タンパク質を親水性 基とした両親媒性榭脂;ポリ乳酸 ポリエチレングリコールブロック共重合体、ポリ ε —力プロラタトン—ポリエチレングリコールブロック共重合体、ポリリンゴ酸—ポリリンゴ 酸アルキルエステルブロック共重合体などの両親媒性榭脂などが挙げられる力 これ らに限定されない。  [0058] It is preferable that the resin is composed of an amphiphilic polymer in the culture substrate according to this embodiment. In the present 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. Combined 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 ε-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.
[0059] 他の実施形態にお!ヽて、本発明に係る培養基材は、複数の孔を有して!/ヽることが 好ましい。本実施形態に係る培養基材において、上記孔は、貫通孔または非貫通孔 のいずれであってもよぐ少なくとも表面部に多孔構造を有していればよい。また本実 施形態に係る培養基材においては、上記複数の孔の各々が基材内部において連通 して 、る「連続性多孔構造」であることがより好ま 、。 [0060] 本実施形態に係る培養基材にお!/、て、上記孔の平均孔径は、 0. 1〜20 μ mであ ることが好ましい。また、本実施形態に係る培養基材において、上記孔の開口形状は 特に限定されず、円形状、楕円形状、正方形状、長方形状、六角形状などのいかな る形状であってもよい。 [0059] In other embodiments, the culture substrate according to the present invention preferably has a plurality of pores. In the culture substrate according to the present embodiment, 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. Further, in the culture substrate according to this embodiment, it is more preferable that each of the plurality of pores has a “continuous porous structure” in which the inside of the substrate communicates. [0060] In the culture substrate according to the present embodiment, it is preferable that the average pore diameter of the pores is 0.1 to 20 µm. Further, in the culture substrate according to the present embodiment, 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.
[0061] 本明細書中において使用される場合、用語「孔径」は、孔の開口形状に対する最 大内接円の直径が意図され、例えば、孔の開口形状が実質的に円形状である場合 はその円の直径が意図され、実質的に楕円形状である場合はその楕円の短径が意 図され、実質的に正方形状である場合はその正方形の辺の長さが意図され、実質的 に長方形状である場合はその長方形の短辺の長さが意図される。  [0061] As used herein, 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.
[0062] さらに、本実施形態に係る培養基材において、上記孔の孔径の変動係数〔=標準 偏差 ÷平均値 X 100 (%)〕は 30%以下であることが好ましい。  [0062] Further, in the culture substrate according to the present embodiment, the pore diameter variation coefficient [= standard deviation ÷ average value X 100 (%)] is preferably 30% or less.
[0063] なおさらに、本実施形態に係る培養基材において、幹幅が 0. 01〜7 μ mであること が好ましい。本明細書中において使用される場合、「幹幅」は、孔と孔との間の幅が 意図される。  [0063] Furthermore, in the culture substrate according to the present embodiment, it is preferable that the stem width is 0.01 to 7 μm. As used herein, “stem width” is intended to be the width between holes.
[0064] 本実施形態に係る培養基材において、上記複数の孔は規則的に配列されているこ とが好ましぐより好ましくは、該複数の孔はハ-カム様に配列されている。本明細書 中において使用される場合、用語「ノヽ二カム様 (ノヽ二カム様構造)」は、孔径がほぼ一 定の複数の孔が規則正しく蜂巣状に配列してなる多孔構造が意図される。  [0064] In the culture substrate according to the present embodiment, it is preferable that the plurality of holes are regularly arranged, and more preferably, the plurality of holes are arranged in a Harkham-like manner. As used herein, 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. .
[0065] ハ-カム様構造体の製造方法としては、特許文献 2に記載の方法以外に、ナノイン プリントを含む金型技術 (サブミクロン〜 100ミクロン程度の均一な空孔を持つハ-カ ム構造体を得る技術)、コロイド微粒子分散液を乾燥させ、集積したコロイド結晶を铸 型としてハ-カム状の多孔質膜を得る方法などが知られている。しかし、前者の方法 では、铸型を剥離する際に空孔の形状が崩れるため、原理的に铸型の構造を正確 に反映させることが困難であり、後者の方法では、集積に時間が力かること、材料を 流し込んだ後に铸型を除去しなければならないことなどの問題を有する。よって、多 孔構造が規則正しく配列するハニカム様構造体の簡便な作製方法は、特許文献 2に 記載される方法が最も好ま Uヽが、本発明に係る培養基材を作製するための方法は 、これに限定されない。 [0066] 本発明に係る培養基材を用いる対象は、幹細胞であれば特に限定されず、神経幹 細胞、造血幹細胞、間葉系幹細胞、体性幹細胞または胚性幹細胞のいずれであつ てもよい。 [0065] In addition to the method described in Patent Document 2, 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. However, in the former method, the shape of the voids collapses when the saddle is peeled off, so in principle it is difficult to accurately reflect the saddle structure. In the latter method, 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. [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. .
[0067] 再生医療に応用される組織幹細胞としては、すでに実用化されている造血幹細胞（ 造血器悪性腫瘍、造血不全、免疫不全、代謝性疾患、固形腫瘍など）、実用化間近 と考えられている間葉系幹細胞 (骨折後の骨再生、筋疾患、虚血部欠陥新生など） および神経幹細胞 (末梢神経損傷 (外傷)、虚血、神経系悪性腫瘍、神経変性疾患 など）、ならびに基礎研究の段階である肝幹細胞 (肝不全)、筋幹細胞 (筋疾患）、脾 幹細胞 (糖尿病)、皮膚幹細胞 (熱傷、皮膚切除後）、網膜幹細胞 (網膜変性疾患)、 および毛包幹細胞 (脱毛症）が挙げられる (括弧内に主な対象疾患を示す)。  [0067] As tissue stem cells applied to regenerative medicine, hematopoietic stem cells (hematopoietic malignant tumor, hematopoietic insufficiency, immunodeficiency, metabolic disease, solid tumor, etc.) already put into practical use are considered to be in practical use. Mesenchymal stem cells (bone regeneration after fracture, muscle disease, ischemic defect, etc.) and 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).
[0068] 従来、神経変性疾患に対する遺伝子治療は、非分裂細胞であるニューロンに遺伝 子を導入し得るアデノウイルスベクターまたはレンチウィルスベクターを用いた研究に よってその可能性が示唆されて 、る。神経幹細胞に遺伝子導入して細胞移植すれ ば、ウィルスベクター特有の危険性を完全に排除することができ、ゲノムに遺伝子導 入された細胞のクローンを安全に取得することができ、その結果、所望の遺伝子を発 現する細胞を所望の部位に所望の数だけ移植することができる。  [0068] Conventionally, gene therapy for neurodegenerative diseases has been suggested by research using adenovirus vectors or lentiviral vectors that can introduce genes into neurons that are non-dividing cells. By introducing a gene into a neural stem cell and transplanting the cell, it is possible to completely eliminate the risks inherent in viral vectors, and it is possible to safely obtain a clone of a cell that has been introduced into the genome. A desired number of cells expressing these genes can be transplanted to a desired site.
[0069] 神経幹細胞を用いる治療対象であることが想定されている疾患としては、パーキン ソン病、筋萎縮性側索硬化症、多発性硬化症、脳梗塞などが挙げられ、これらの疾 患に対する治療標的部位は、それぞれ中脳黒質ドーパミンニューロン、運動ニューロ ン、オリゴデンドロサイト、ニューロンおよび Zまたはグリアである。このように特定の疾 患を処置する場合は、特定の細胞を標的とすればよいので、ウィルスベクターを用い るよりも神経幹細胞を用いる方が好まし 、。  [0069] Diseases assumed to be treatment targets using 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. Thus, when treating a specific disease, it is preferable to use a neural stem cell rather than a viral vector because it is only necessary to target a specific cell.
[0070] なお、本発明の一実施形態である多孔性フィルムを備えた基材を用いて細胞を組 織化すれば、人工臓器に利用することができる。人工臓器として用いる場合は体内 に埋め込むことが必要であるので、本発明に係る基材は長期的には生体内へ吸収さ れることが好ましい。多孔構造を有する従来の基材を用いた場合では、細胞培養に 要する時間内では構造が安定であるが必要な時間が経過した後に分解するという生 体内での用途に好ましいものは存在しない。換言すれば、本発明に係る培養基材を 細胞工学および細胞培養技術と組み合わせて人工臓器等の医療用途へ応用する ためには生分解性材料を用いることが必要とされる。 [0070] It should be noted that if cells are organized using a substrate provided with a porous film according to an embodiment of the present invention, it can be used for an artificial organ. When used as an artificial organ, it is necessary to be embedded in the body, so that the base material according to the present invention is preferably absorbed into the living body in the long term. In the case of using a conventional base material having a porous structure, there is nothing preferable for an in vivo use in which the structure is stable within the time required for cell culture, but decomposes after the necessary time has elapsed. In other words, 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.
[0071] また、本発明に係る培養基材を用いれば、細胞を無血清にて培養することができる ので、自家移植に利用するための所望の細胞を安全に供給することができる。本発 明に係る培養基材を用いれば、増殖因子を用いることなく細胞を培養することができ るので、所望の細胞を低コストにて供給することができる。  [0071] Furthermore, if the culture substrate according to the present invention is used, 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.
[0072] つまり、本発明の目的は、幹細胞を分化させることなく増殖させるための培養基材 を提供することにあるのであって、本明細書中に具体的に記載した薄膜の製造方法 、薄膜の厚さ、榭脂成分、孔の数および深さ、孔の形状等の条件に存するのではな い。したがって、上記方法以外を用いて製造された培養基材もまた本発明の範囲に 属することに留意しなければならない。  [0072] That is, 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.
[0073] (2)幹細胞分化用培養基材  [0073] (2) Culture substrate for stem cell differentiation
本発明は、幹細胞を分化させるための培養基材を提供する。一実施形態において 、本発明に係る培養基材は、好ましくは 0. 01〜： LOO mの範囲の膜厚を有している 薄膜を備えている。本実施形態に係る培養基材において、上記薄膜は 1層であって も複数積層されていてもよぐ基板 (例えば、プラスチック、ガラスなど)上に該薄膜が 積層されていてもよい。  The present invention provides a culture substrate for differentiating stem cells. In one embodiment, 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. In the culture substrate according to this embodiment, 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.
[0074] 本実施形態に係る培養基材にお!/、て、上記薄膜は榭脂からなることが好ま 、。本 実施形態に係る培養基材において、上記榭脂としては、特に限定されないが、培養 用途であることを考慮すると毒性の少ないものが好ましい。また、本実施形態に係る 培養基材を、特許文献 2に記載されるような方法に従って作製する場合は、有機溶 媒に溶解する高分子化合物 (ポリマー)であることが好ましい。本実施形態に係る培 養基材がポリマーカゝらなる薄膜を備えている場合、高分子薄膜を形成するために、ィ ンクジェット方式またはスクリーン方式のような印刷法を用いて高分子を所望の形状 およびサイズにペーストもよいし、フォトリソグラフィ一法などを用いてさらに表面を整 形してちょい。  [0074] In the culture substrate according to this embodiment, it is preferable that the thin film is made of coconut. In the culture substrate according to the present embodiment, the above-mentioned coagulant is not particularly limited, but considering that it is used for culture, one having less toxicity is preferable. Further, when 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. In the case where the culture substrate according to the present embodiment includes a thin film made of a polymer cover, 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.
[0075] なお、このような手法を用いてポリマー力もなる薄膜を形成する場合は、支持体 (基 板）が必要であるが、基板としては、寸度的に安定なものであれば特に限定されず、 例えば、紙、プラスチック（例えば、ポリエチレン、ポリプロピレン、ポリスチレン等）がラ ミネートされた紙、金属板 (例えば、アルミニウム、亜鉛、銅等）、プラスチックフィルム（ 例えば、二酢酸セルロース、三酢酸セルロース、プロピオン酸セルロース、酪酸セル ロース、酢酸酪酸セルロース、硝酸セルロース、ポリエチレンテレフタレート、ポリェチ レン、ポリスチレン、ポリプロピレン、ポリカーボネート、ポリビュルァセタール等）等が 挙げられる。これらは、榭脂フィルムまたは金属板などの単一成分のシートであっても[0075] When a thin film having a polymer force is formed using such a method, a support (substrate) is required, but the substrate is not particularly limited as long as it is dimensionally stable. not, For example, paper, paper laminated with plastic (eg, polyethylene, polypropylene, polystyrene, etc.), metal plate (eg, aluminum, zinc, copper, etc.), plastic film (eg, cellulose diacetate, cellulose triacetate, propionic acid) Cellulose, butyric acid cellulose, cellulose acetate butyrate, cellulose nitrate, polyethylene terephthalate, polyethylene, polystyrene, polypropylene, polycarbonate, polybulucetal, etc.). These can be single component sheets such as resin films or metal plates
、 2以上の材料の積層体であってもよい。 A laminate of two or more materials may be used.
このようなポリマーとしては、ポリブタジエン、ポリイソプレン、スチレン ブタジエン 共重合体、アクリロニトリル—ブタジエン—スチレン共重合体などの共役ジェン系高 分子；ポリ ε一力プロラタトン；ポリウレタン；酢酸セルロース、セルロイド、硝酸セル口 ース、ァセチルセルロース、セロファンなどのセルロース系高分子；ポリアミド 6、ポリア ミド 66、ポリアミド 610、ポリアミド 612、ポリアミド 12、ポリアミド 46などのポリアミド系高 分子；ポリテトラフルォロエチレン、ポリトリフルォロエチレン、パーフルォロエチレン プロピレン共重合体などのフッ素系高分子；ポリスチレン、スチレン エチレン プロ ピレン共重合体、スチレン一エチレン一ブチレン共重合体、スチレン一イソプレン共 重合体、塩素化ポリエチレン—アクリロニトリル スチレン共重合体、メタクリル酸エス テル スチレン共重合体、スチレン—アクリロニトリル共重合体、スチレン 無水マレ イン酸共重合体、アクリル酸エステル—アクリロニトリル スチレン共重合体などのス チレン系高分子；ポリエチレン、塩素化ポリエチレン、エチレン aーォレフイン共重 合体、エチレン 酢酸ビュル共重合体、エチレン一塩化ビュル共重合体、エチレン 酢酸ビュル共重合体、ポリプロピレン、ォレフィン ビュルアルコール共重合体、 ポリメチルペンテンなどのォレフィン系高分子;フエノール榭脂、アミノ榭脂、尿素榭 脂、メラミン榭脂、ベンゾグアナミン榭脂などのホルムアルデヒド系高分子;ポリブチレ ンテレフタレート、ポリエチレンテレフタレート、ポリエチレンナフタレートなどのポリェ ステル系高分子;エポキシ榭脂；ポリ（メタ)アクリル酸エステル、ポリ— 2—ヒドロキシェ チルアタリレート、メタクリル酸エステル 酢酸ビュル共重合体などの（メタ）アクリル系 高分子;ノルボルネン系榭脂；シリコン榭脂；ポリ乳酸、ポリヒドロキシ酪酸、ポリグリコ ール酸などのヒドロキシカルボン酸の重合体などが挙げられ、これらは単独で使用さ れても組み合わせて使用されてもょ 、。 Examples of such polymers 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, methacrylate ester styrene copolymer, styrene-acrylonitrile copolymer, styrene maleic anhydride copolymer, acrylate ester-acrylonitrile styrene copolymer; polyethylene Olefins such as chlorinated polyethylene, ethylene a-olefin copolymer, ethylene acetate butyl copolymer, ethylene monochloride butyl copolymer, ethylene acetate butyl copolymer, polypropylene, olefin fin butyl alcohol copolymer, polymethylpentene, etc. Polymers: Formaldehyde 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.
[0077] なお、本発明に係る培養基材を構成するポリマーは、非生分解性榭脂であっても 生分解性榭脂であってもよ!、が、生体外で幹細胞のインビトロ増幅を目的とした培養 を行う場合は、生分解性である必要はない。また、培養基材の効果を生体内で長期 間持続させることが好ましい場合は、非生分解性榭脂を用いればよぐ生体で長期 間持続させることが好ましくない場合は、生分解性榭脂を用いればよい。好ましい生 分解性榭脂としては、ポリ乳酸、ポリ —力プロラタトン）、およびポリ（グリコール酸 —乳酸)共重合体が挙げられ、好ましい非生分解性榭脂としては、ポリブタジエン、 ポリウレタン、およびポリ（メタ）アタリレートが挙げられる。  [0077] 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.
[0078] 本実施形態に係る培養基材にお!/ヽて、上記樹脂が両親媒性のポリマーを含んで 構成されることが好ましい。本実施形態において、好ましい両親媒性ポリマーとして は、ポリエチレングリコール zポリプロピレングリコールブロック共重合体；アクリルアミ ドポリマーを主鎖骨格とし疎水性側鎖としてドデシル基と親水性側鎖としてラタトース 基またはカルボキシル基を併せ持つ両親媒性榭脂；へノ リンゃデキストラン硫酸、核 酸（DNAや RNA)などのァ-オン性高分子と長鎖アルキルアンモ-ゥム塩とのィォ ンコンプレックス；ゼラチン、コラーゲン、アルブミンなどの水溶性タンパク質を親水性 基とした両親媒性榭脂;ポリ乳酸 ポリエチレングリコールブロック共重合体、ポリ ε —力プロラタトン—ポリエチレングリコールブロック共重合体、ポリリンゴ酸—ポリリンゴ 酸アルキルエステルブロック共重合体などの両親媒性榭脂などが挙げられる力 これ らに限定されない。  [0078] In the culture substrate according to the present embodiment, it is preferable that the resin includes an amphiphilic polymer. In the present 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. Combined 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 ε-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.
[0079] 別の実施形態において、本発明に係る培養基材は、複数の孔を有していることが 好ましい。本実施形態に係る培養基材において、上記孔は、貫通孔または非貫通孔 のいずれであってもよぐ少なくとも表面部に多孔構造を有していればよい。また本実 施形態に係る培養基材においては、上記複数の孔の各々が基材内部において連通 して 、る「連続性多孔構造」であることがより好ま 、。  [0079] In another embodiment, the culture substrate according to the present invention preferably has a plurality of pores. In the culture substrate according to the present embodiment, 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. Further, in the culture substrate according to this embodiment, it is more preferable that each of the plurality of pores has a “continuous porous structure” in which the inside of the substrate communicates.
[0080] 本実施形態に係る培養基材にお!/、て、上記孔の平均孔径は、 0. 1〜20 μ mであ ることが好ましい。また、本実施形態に係る培養基材において、上記孔の開口形状は 特に限定されず、円形状、楕円形状、正方形状、長方形状、六角形状などのいかな る形状であってもよい。 [0080] In the culture substrate according to the present embodiment, it is preferable that the average pore diameter of the pores is 0.1 to 20 µm. Further, in the culture substrate according to the present embodiment, 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.
[0081] 本明細書中において使用される場合、用語「孔径」は、孔の開口形状に対する最 大内接円の直径が意図され、例えば、孔の開口形状が実質的に円形状である場合 はその円の直径が意図され、実質的に楕円形状である場合はその楕円の短径が意 図され、実質的に正方形状である場合はその正方形の辺の長さが意図され、実質的 に長方形状である場合はその長方形の短辺の長さが意図される。  [0081] As used herein, 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.
[0082] さらに、本実施形態に係る培養基材において、上記孔の孔径の変動係数〔=標準 偏差 ÷平均値 X 100 (%)〕は 30%以下であることが好ましい。  [0082] Further, in the culture substrate according to the present embodiment, the pore diameter variation coefficient [= standard deviation ÷ average value X 100 (%)] is preferably 30% or less.
[0083] なおさらに、本実施形態に係る培養基材において、幹幅が 0. 01〜7 μ mであること が好ましい。本明細書中において使用される場合、「幹幅」は、孔と孔との間の幅が 意図される。  [0083] Still further, in the culture substrate according to the present embodiment, the stem width is preferably 0.01 to 7 µm. As used herein, “stem width” is intended to be the width between holes.
[0084] 本実施形態に係る培養基材において、上記複数の孔は規則的に配列されているこ とが好ましぐより好ましくは、該複数の孔はハ-カム様に配列されている。本明細書 中において使用される場合、用語「ノヽ二カム様 (ノヽ二カム様構造)」は、孔径がほぼ一 定の複数の孔が規則正しく蜂巣状に配列してなる多孔構造が意図される。  [0084] In the culture substrate according to the present embodiment, it is preferable that the plurality of holes are regularly arranged, and more preferably, the plurality of holes are arranged in a Harkham-like manner. As used herein, 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. .
[0085] ハ-カム様構造体の製造方法としては、特許文献 2に記載の方法以外に、ナノイン プリントを含む金型技術 (サブミクロン〜 100ミクロン程度の均一な空孔を持つハ-カ ム構造体を得る技術)、コロイド微粒子分散液を乾燥させ、集積したコロイド結晶を铸 型としてハ-カム状の多孔質膜を得る方法などが知られている。しかし、前者の方法 では、铸型を剥離する際に空孔の形状が崩れるため、原理的に铸型の構造を正確 に反映させることが困難であり、後者の方法では、集積に時間が力かること、材料を 流し込んだ後に铸型を除去しなければならないことなどの問題を有する。よって、多 孔構造が規則正しく配列するハニカム様構造体の簡便な作製方法は、特許文献 2に 記載される方法が最も好ま Uヽが、本発明に係る培養基材を作製するための方法は 、これに限定されない。  [0085] In addition to the method described in Patent Document 2, 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. However, in the former method, the shape of the voids collapses when the saddle is peeled off, so in principle it is difficult to accurately reflect the saddle structure. In the latter method, 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.
[0086] 本発明に係る培養基材を用いる対象は、幹細胞であれば特に限定されず、神経幹 細胞、造血幹細胞、間葉系幹細胞、体性幹細胞または胚性幹細胞のいずれであつ てもよい。 [0087] また、本発明に係る培養基材を用いれば、細胞を無血清にて培養することができる ので、自家移植に利用するための所望の細胞を安全に供給することができる。本発 明に係る培養基材を用いれば、分化誘導因子を用いることなく細胞を培養することが できるので、所望の細胞を低コストにて供給することができる。 [0086] 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. . [0087] Furthermore, if the culture substrate according to the present invention is used, 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.
[0088] つまり、本発明の目的は、幹細胞を分化させるための培養基材を提供することにあ るのであって、本明細書中に具体的に記載した薄膜の製造方法、薄膜の厚さ、榭脂 成分、孔の数および深さ、孔の形状等の条件に存するのではない。したがって、上記 方法以外を用いて製造された培養基材もまた本発明の範囲に属することに留意しな ければならない。  That is, 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.
[0089] 尚、発明を実施するための最良の形態の項においてなした具体的な実施態様およ び以下の実施例は、あくまでも、本発明の技術内容を明らかにするものであって、そ のような具体例にのみ限定して狭義に解釈されるべきものではなぐ当業者は、本発 明の精神および添付の請求の範囲内で変更して実施することができる。  [0089] It should be noted that the specific embodiments made in the section of the best mode for carrying out the invention and the following examples are merely to clarify the technical contents of the present invention. Those skilled in the art who are not to be construed as limited to the specific examples as described above can make various modifications within the spirit of the present invention and the scope of the appended claims.
[0090] また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中に ぉ ヽて参考として援用される。  [0090] In addition, all the academic literatures and patent literatures described in this specification are incorporated herein by reference in their entirety.
[0091] 〔実施例〕  [0091] [Example]
〔1 :細胞培養基板の作製〕  [1: Production of cell culture substrate]
〔1. 1 :自己組織ィ匕多孔フィルムおよび平膜の作製〕  [1.1: Preparation of self-assembled porous film and flat film]
ポリ 一力プロラタトン）（Poly( ε -caprolactone)； (株）和光純薬工業、 MW70 , 000-100, 000 :以下 PCLと称する）と両親媒性アクリルアミドポリマー（Cap)とを 重量比 10 : 1の割合で混合した後にクロ口ホルムに溶解し、濃度を lOmgZmLに調 製した。多孔フィルムを、ガラスシャーレ (直径 9cm)にて作製した高分子混合溶液を キャストして高湿度雰囲気下にて作製した。多孔フィルムの孔径は、キャストする高分 子溶液の量を変えることにより制御することができた。各フィルムの孔径、幹径および 空孔率を、走査型電子顕微鏡 (SEM) (HITACHI, S— 3500)を用いて測定した。 画像 1枚あたり 5個の孔を選択してその直径の平均を孔径として求め、合計 5枚の画 像にっ 、て孔径を測定した。多孔フィルムの幹幅の最も細 、部分を幹径として測定 し、画像解析用ソフトウェア Scion Image (Scion Corporation)を用いて空孔率 を測定した（図 1)。また PCL平膜を、上記混合溶液を 18mm角のカバーガラス上に 滴下し、 1000rpm、 30秒の条件でスピンコーター（MIKAS A)を用いて作製した。 Poly (ε-caprolactone); Wako Pure Chemical Industries, Ltd. (MW70, 000-100, 000: hereinafter referred to as PCL) and amphiphilic acrylamide polymer (Cap) in a weight ratio of 10: 1 After mixing at a ratio of 0.5, dissolved in black mouth form, the concentration was adjusted to 10 mgZmL. A porous film was prepared in a high humidity atmosphere by casting a polymer mixed solution prepared in a glass petri dish (diameter 9 cm). The pore size of the porous film could be controlled by changing the amount of polymer solution to be cast. The hole diameter, trunk diameter and porosity of each film were measured using a scanning electron microscope (SEM) (HITACHI, S-3500). Five holes per image were selected and the average of the diameters was determined as the hole diameter, and the hole diameter was measured using a total of five images. The narrowest part of the trunk width of the porous film is measured as the trunk diameter, and the porosity is measured using the image analysis software Scion Image (Scion Corporation). Was measured (Fig. 1). 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.
[0092] 〔2：細胞培養基板処理〕  [0092: Cell processing substrate treatment]
作製した自己組織ィ匕多孔フィルムを切り取り、 18mm角のカバーガラス（MATSU NAMI)に密着させた。 PCL多孔フィルムおよび PCL平膜を、 1 プロパノール (Wa ko)中で 5分間浸漬して洗浄し、細胞培養容器 35mmZnon— treated polystyle ne culture dish (IWAKI)中にてエタノールおよび UV照射によって滅菌した後、 Poly(L— Lysin)溶液（50mgZl Poly (L— Lysine) (Sigma)、 0. 1M ホウ酸（W ako) (pH8. 3) )に 1時間浸漬した後、滅菌水で 3回洗浄し、さらに FBS (Fetal Bov ine Serum)を含む培地（Opti—MEM、 10% FBS)中にて 37°Cで 1時間インキ ュペートしてコンディショニングを行った後に細胞培養に供した。  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.
[0093] 〔3 :神経細胞調製〕  [0093] [3: Neural cell preparation]
神経細胞を、胎生 14日目の ICRマウスの大脳皮質組織から、以下のように調製し た。まず、妊娠 14日目のマウス力も胎仔を取り出した後に脳を摘出した。さらに、大 脳半球から大脳皮質を分離して培地中（Opti-MEM、 Gibco)に回収し、パスツー ルピペットによって細胞を分散させた。次いで、血球計算板を用いて細胞数を計数し 、トリパンブルー（Gibco)染色による Viability測定を行った。  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.
[0094] 〔4 :神経細胞培養〕  [4: Nerve cell culture]
マウス胎仔大脳皮質組織力 調製した細胞懸濁液を、細胞密度 2. 0 104細胞7 cm2になるように培養基板上に播種した。 37°C、 5%COの条件下にて、 1日目は血 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
2  2
清培地（Opti— MEM、 10%FBS、 2— Mercaptoethanol(Gibco) )、 2日目以降 は無血清培地（Opti— MEM、 B27 Supplement (Gibco)、 2-Mercaptoethan ol)を用いて培養した。 5日間培養した後、走査型電子顕微鏡および共焦点レーザ 一顕微鏡観察（OLYMPUS、 FLUOVIEW FV300)を用いて細胞形態および突 起伸展の様子を観察した。  Culture was performed using clear medium (Opti-MEM, 10% FBS, 2-Mercaptoethanol (Gibco)), and serum-free medium (Opti-MEM, B27 Supplement (Gibco), 2-Mercaptoethanol) on and after the second day. After culturing for 5 days, the cell morphology and the state of protruding extension were observed using a scanning electron microscope and confocal laser single microscope observation (OLYMPUS, FLUOVIEW FV300).
[0095] [5：走査型電子顕微鏡観察〕 [0095] [5: Scanning electron microscope observation]
5日間培養した神経細胞を PBSで洗浄し、 2. 5%ダルタールアルデヒド ZPBSを 用 ヽて 40Cでー晚固 した。 ヽで、 PBS、 900/0PBS、 70%PBS, 50%PBS, 30 %PBS、および MilliQ水で洗浄し、エタノール（20%、 50%、 70%、 99%)で順次 脱水した後 3時間減圧乾燥した。乾燥させた試料にイオンスパッタリング装置 (HITA CHI, E- 1030)を用いて白金パラジウムを蒸着させ SEMを用いて観察した。 5 days cultured neurons were washed with PBS, and 4 0 C De晚固Te useヽa 2.5% Dar glutaraldehyde ZPBS. In ヽ, PBS, 90 0/0 PBS , 70% PBS, 50% PBS, 30 It was washed with% PBS and MilliQ water, dehydrated successively with ethanol (20%, 50%, 70%, 99%) and then dried under reduced pressure for 3 hours. Platinum palladium was vapor-deposited on the dried sample using an ion sputtering apparatus (HITA CHI, E-1030) and observed using SEM.
[0096] [6：共焦点レーザー顕微鏡観察〕 [0096] [6: Observation with confocal laser microscope]
[6. 1 : j8 Tubulinlll免疫化学染色〕  [6.1: j8 Tubulinlll immunochemical staining]
5日間培養した神経細胞を PBSで洗浄し、 4%パラホルムアルデヒド ZPBSを添カロ して、室温で 1時間放置して細胞を固定した。 PBSで 3回（各 10分間ずつ）洗浄した 後、 Blocking solution (PBS中 5%ャギ血清、 2. 5% BSA、 0. 2% Triton— X Neurons cultured for 5 days were washed with PBS, supplemented with 4% paraformaldehyde ZPBS, and left at room temperature for 1 hour to fix the cells. After washing 3 times with PBS (10 minutes each), Blocking solution (5% goat serum in PBS, 2.5% BSA, 0.2% Triton— X
100)を添加して、室温で 1時間細胞をインキュベートした。 Blocking solutionを 除去して、細胞を、一次抗体 (抗 j8— TubulinIII (PBSにて 1 : 800) )とともに室温で 1時間インキュベートした。 PBSで 3回（各 10分間ずつ）洗浄した後、細胞を、 2次抗 体 (FITC複合体化マウス IgG (PBSにて 1 :400)とともに室温で 1時間インキュベート した。 PBSで 3回 (各 10分間ずつ）、蒸留水で 1回洗浄した後、サンプルをスライドガ ラスに載せ Mounting media (KPL)によってマウントした。 100) was added and the cells were incubated for 1 hour at room temperature. The blocking solution was removed, and the cells were incubated with the primary antibody (anti-j8-Tubulin III (1: 800 in PBS)) for 1 hour at room temperature. After washing with PBS 3 times (10 minutes each), cells were incubated with secondary antibody (FITC-conjugated mouse IgG (1: 400 in PBS) for 1 hour at room temperature. 3 times with PBS (each After washing once with distilled water (10 minutes each), the sample was placed on a slide glass and mounted with Mounting media (KPL).
[0097] 〔6. 2 :Nestin免疫化学染色〕 [0097] [6.2: Nestin immunochemical staining]
5日間培養した神経細胞を PBSで洗浄し、 4%パラホルムアルデヒド ZPBSをカロえ て室温で 1時間放置して細胞を固定した。 PBSで 3回（各 10分間ずつ）洗浄した後、 Blocking solution (PBS中 5%ャギ血清、 2. 5% BSA、 0. 2% Triton— X 1 00)を添加して、室温で 1時間細胞をインキュベートした。 Blocking solutionを除 去して、細胞を、一次抗体 (抗 Nestin抗体 (PBSにて 1 : 1000) )とともに室温で 1時 間インキュベートした。 PBSで 3回（各 10分間ずつ）洗浄した後、細胞を、ピオチンィ匕 抗マウス IgG (PBSにて 1： 1000)とともに室温で 1時間インキュベートした。 PBSで洗 浄した後、細胞を、 Alexa488標識 avidin (PBSにて 1： 2000)とともに 30分間インキ ュペートした。 PBSで 3回（各 10分間ずつ）、蒸留水で 1回洗浄した後、サンプルをス ライドガラスに載せ Mounting media (KPL)によってマウントした。  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. After washing with PBS, cells were incubated with Alexa488 labeled avidin (1: 2000 in PBS) for 30 minutes. After washing with PBS three times (each for 10 minutes) and once with distilled water, the sample was mounted on a slide glass and mounted with Mounting media (KPL).
[0098] 〔6. 3 : BrdU取り込みラベリング〕 [0098] [6.3: BrdU incorporation labeling]
培養培地を 20 μ M BrdUを含む培地と交換して 2時間培養することによって、増 殖している細胞の核内に BrdUを取り込ませた。次いで、 10%ホルマリンを用いて室 温にて 2時間細胞を固定した。 PBSで 3回（各 10分間ずつ）洗浄し、 2M HC1溶液 中にて 37°Cで 60分間インキュベートした後、 0. 1M H BO緩衝液で 2回（5分間ず By replacing the culture medium with a medium containing 20 μM BrdU and culturing for 2 hours, 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)
3 4  3 4
つ）、 PBSで 2回洗浄した。次いで、 Blocking solution (PBS中 5%ャギ血清、 2. 5% BSA、0. 2% Triton -X 100)を添カ卩して、室温で 1時間細胞をインキュべ ートした。 Blocking solutionを除去した後、細胞を、一次抗体（抗 BrdUマウス IgG (PBSにて 1 : 1000)とともに室温で 1時間インキュベートした。 PBSで 3回（各 10分間 ずつ）洗浄した後、細胞を、ピオチンィ匕抗マウス IgG (PBS〖こて 1： 1000)ととも〖こ室温 で 1時間インキュベートした。さらに PBSで洗浄した後、 Alexa488標識 avidin(PBS にて 1 : 2000)で細胞を 30分間インキュベートした。 PBSで 3回（各 10分間）、蒸留水 で 1回洗浄した後、サンプルをスライドガラスに載せ Mounting media (KPL)によつ てマウントした。  And washed twice with PBS. 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. After washing 3 times with PBS (10 minutes each), 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).
[0099] 〔7 :結果〕 [0099] [7: Result]
胎生 14日目のマウス胎仔大脳皮質組織力 調製した細胞中には、多くの神経幹 細胞が存在して 、る。神経幹細胞と平膜または多孔フィルムとの接着による細胞の形 態変化に対する影響および細胞の増殖または分化に対する影響を、 SEMおよび免 疫化学染色による形態観察によって検討した。  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.
[0100] まず、マウス胎仔大脳皮質組織力 調製した細胞を PCL平膜上または多孔フィル ム上に播種し、 37°Cで 4時間インキュベートした後放置した。その後、 Nestinに対す る免疫化学染色および BudUのラベリングを行った。その結果、 Nestin染色および B rdUラベリングは陽性を示し、調製した細胞中には神経幹細胞が多く含まれて 、るこ とが分かった（図 2および 3)。  [0100] First, cells prepared from mouse fetal cerebral cortical tissue strength were seeded on a PCL flat membrane or a porous film, incubated at 37 ° C for 4 hours, and then allowed to stand. Subsequently, immunochemical staining for Nestin and labeling of BudU were performed. As a result, Nestin staining and BrdU labeling were positive, and it was found that the prepared cells contained many neural stem cells (Figs. 2 and 3).
[0101] 次に、 PCL平膜または孔径 3 μ mの PCL多孔フィルム上において、大脳皮質組織 力も調製した細胞を播種し、培養 5日後、同様の実験を行い検討した。また、細胞の 形態を走査型電子顕微鏡、共焦点レーザー顕微鏡で観察した（図 4)。  [0101] Next, cells with a prepared cerebral cortical tissue strength were seeded on a PCL flat membrane or a PCL porous film having a pore size of 3 µm, and the same experiment was conducted after 5 days of culture. Cell morphology was observed with a scanning electron microscope and confocal laser microscope (Fig. 4).
[0102] 平膜上では、神経細胞の形態は紡錘形であり、多数の神経突起が伸展しネットヮ ーク構造を形成していた（図 4a)。一方、孔径 3 mの PCL多孔フィルム上では、神 経細胞は、直径約 30〜50 μ mの球状の凝集体 (スフヱロイド様凝集塊）を形成した。 また神経突起は凝集塊の底部において互いに集合して太い突起となり、凝集体から 5〜7本の突起を放射状に伸展させ、ネットワーク様構造を形成していた（図 4b)。 [0102] On the flat membrane, the form of neurons was spindle-shaped, and many neurites were extended to form a network structure (Fig. 4a). On the other hand, on the PCL porous film having a pore diameter of 3 m, neuronal cells formed spherical aggregates (spheroid-like aggregates) having a diameter of about 30 to 50 μm. Also, the neurites gather together at the bottom of the agglomerate to form thick protrusions, Five to seven protrusions were radially extended to form a network-like structure (Fig. 4b).
[0103] さらに、 PCL平膜または孔径 3 m、 5 m、 8 m、 10 mの PCL多孔フィルム上 に大脳皮質組織力ゝら調製した細胞を播種し、培養 3〜7日後、細胞の形態を走査型 電子顕微鏡にて、 β - tubulin IIIおよび Nestinによる免疫化学染色を共焦点レー ザ一顕微鏡にて観察した。  [0103] Furthermore, cells prepared by cerebral cortical tissue strength were seeded on a PCL flat membrane or a PCL porous film having a pore size of 3 m, 5 m, 8 m, or 10 m, and after 3-7 days in culture, the cell morphology was changed. Immunochemical staining with β-tubulin III and Nestin was observed with a confocal laser microscope using a scanning electron microscope.
[0104] 平膜上では、神経細胞のマーカーである β - tubulin ΠΙによる免疫化学染色は 陽性を示した（図 5)。また、 Nestin染色および BrdUラベリングは陰性を示した（図 1 0および 11)。このことより、神経幹細胞は平膜上においては、未分化な神経細胞か ら成熟した神経細胞になるということが示された。一方、孔径 の PCL多孔フィル ム上では、神経突起において、神経細胞のマーカーである 13 - tubulin ΠΙによる免 疫化学染色は陽性であり（図 6)、 Nestin染色および BrdUラベリングもまた陽性を示 した（図 12〜14)。し力し、孔径 5 m、 8 m、 10 mの PCL多孔フィルム上では、 ほとんどの細胞が j8 - tubulin ΠΙ陽性であつたが（図 7〜9)、 Nestin陰性であった (図 15)。また、神経細胞の形態については、細胞同士が凝集する力または単独で、 ラメラ体を伸展させて接着する細胞、またはラメラ体を伸展させずに単独でパターン の幹に接着する細胞が観察され、多孔フィルムにおける孔径および幹幅が大きくなる に伴い、細胞形態は、平膜上の形態に類似する傾向が見られた（図 7〜9)。また、 S EM像と β - tubulin ΠΙ蛍光像から、神経突起が多孔フィルムの幹を介して伸展し 、ネットワーク構造を形成して 、ることがわ力つた（図 7〜9)。  [0104] On the flat membrane, immunochemical staining with β-tubulin 神 経, a marker for nerve cells, was positive (Fig. 5). Nestin staining and BrdU labeling were negative (Figures 10 and 11). This indicates that neural stem cells change from undifferentiated neurons to mature neurons on the flat membrane. On the other hand, on PCL porous film with pore size, immunochemical staining with 13-tubulin 免, a neuronal marker, was positive in neurites (Fig. 6), and Nestin staining and BrdU labeling were also positive. (Figures 12-14). However, on PCL porous films with pore sizes of 5 m, 8 m, and 10 m, most cells were positive for j8-tubulin® (Figs. 7-9) but Nestin-negative (Fig. 15). In addition, regarding the morphology of the nerve cells, the cell agglutinates or alone, cells that adhere by extending the lamellae, or cells that adhere to the trunk of the pattern alone without extending the lamellae, are observed, As the pore size and stem width in the porous film increased, the cell morphology tended to resemble that on the flat membrane (FIGS. 7-9). In addition, it was found from the SEM image and β-tubulin fluorescence image that the neurites extended through the trunk of the porous film to form a network structure (FIGS. 7 to 9).
[0105] これらの結果より、神経幹細胞は、孔径 3 μ mの多孔フィルム上においては、比較 的未分化状態を維持すると同時に、自己増殖し、スフエロイド様凝集塊を形成するこ と、および、底部に接している幼若な神経細胞は、序々に成熟した細胞となり神経突 起を放射状に伸展させることがわ力つた。一方、 PCL平膜、または孔径 5 mまたは それ以上（8 m、 10 m)の多孔フィルム上においては、神経幹細胞は、分化して、 基板の形状に従った接着形態を示すことがわ力つた。  [0105] From these results, neural stem cells maintained a relatively undifferentiated state on a porous film with a pore size of 3 μm, 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. On the other hand, on 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. .
産業上の利用の可能性  Industrial applicability
[0106] 本発明に係る培養基材を用いれば、細胞の形態を自由に制御することができる。ま た、本発明に係る培養基材を用いれば、細胞を無血清にて培養することができるの で、自家移植に利用するための所望の細胞を安全に供給することができる。本発明 に係る培養基材を用いれば、増殖因子、分化誘導因子を用いることなく細胞を培養 することができるので、所望の細胞を低コストにて供給することができる。本発明に係 る培養基材を用いれば、特に、自己増幅させることが困難であった幹細胞を分化さ せることなく増殖させることができる。よって、本発明は、遺伝子治療、臓器移植、骨 髄移植、ガン治療、または再生医学といった多岐にわたる医療分野において非常に 有用である。 [0106] If 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.

Claims

請求の範囲 The scope of the claims
[I] 幹細胞を分化させることなく増殖させるための培養基材。  [I] A culture substrate for proliferating stem cells without differentiation.
[2] 0. 01〜： LOO /z mの範囲の膜厚を有している薄膜を備えていることを特徴とする請 求の範囲 1に記載の培養基材。  [2] 0.01-: The culture substrate according to claim 1, comprising a thin film having a film thickness in the range of LOO / zm.
[3] 前記薄膜を複数積層して備えていることを特徴とする請求の範囲 2に記載の培養 基材。 [3] The culture substrate according to claim 2, comprising a plurality of the thin films stacked.
[4] 前記薄膜が榭脂からなることを特徴とする請求の範囲 2に記載の培養基材。  [4] The culture substrate according to claim 2, wherein the thin film is made of rosin.
[5] 前記樹脂が生分解性ポリマーを含んでいることを特徴とする請求の範囲 4に記載の 培養基材。 [5] The culture substrate according to claim 4, wherein the resin contains a biodegradable polymer.
[6] 前記樹脂が両親媒性ポリマーをさらに含んでいることを特徴とする請求の範囲 5に 記載の培養基材。  6. The culture substrate according to claim 5, wherein the resin further contains an amphiphilic polymer.
[7] 前記樹脂が生分解性ポリマーおよび両親媒性ポリマーからなることを特徴とする請 求の範囲 4に記載の培養基材。  [7] The culture substrate according to claim 4, wherein the resin comprises a biodegradable polymer and an amphiphilic polymer.
[8] 前記生分解性ポリマー力 ポリ乳酸、ポリ（ ε—力プロラタトン）、およびポリ（グリコー ル酸ー乳酸)共重合体からなる群より選択されることを特徴とする請求の範囲 5または[8] The biodegradable polymer force selected from the group consisting of polylactic acid, poly (ε-force prolatatone), and poly (glycolic acid-lactic acid) copolymer.
7に記載の培養基材。 8. The culture substrate according to 7.
[9] 前記両親媒性ポリマーが、疎水性側鎖としてドデシル基を有し親水性側鎖としてラ クトース基またはカルボキシル基を有して 、る、アクリルアミドポリマーを主鎖骨格とす る両親媒性榭脂;ポリエチレングリコール系共重合体;および、ァ-オン性高分子と長 鎖アルキルアンモ-ゥム塩とのポリイオンコンプレックス力 なる群より選択されること を特徴とする請求の範囲 6または 7に記載の培養基材。  [9] The amphiphilic polymer having a dodecyl group as a hydrophobic side chain and a lactose group or a carboxyl group as a hydrophilic side chain and having an acrylamide polymer as a main chain skeleton. Claim 6 or 7 characterized in that it is selected from the group consisting of a resin; a polyethylene glycol copolymer; and a polyion complex force of a ionic polymer and a long-chain alkyl ammonium salt. The culture substrate as described.
[10] 複数の孔を有して 、ることを特徴とする請求の範囲 1に記載の培養基材。 [10] The culture substrate according to claim 1, having a plurality of holes.
[I I] 前記複数の孔がハ-カム様に配列されていることを特徴とする請求の範囲 10に記 載の培養基材。  [I I] The culture substrate according to claim 10, wherein the plurality of holes are arranged in a Herkam-like manner.
[12] 各孔が貫通していることを特徴とする請求の範囲 10に記載の培養基材。  [12] The culture substrate according to claim 10, wherein each hole penetrates.
[13] 各孔が連通していることを特徴とする請求の範囲 10に記載の培養基材。 [13] The culture substrate according to claim 10, wherein each hole is in communication.
[14] 前記幹細胞が、神経幹細胞、造血幹細胞、間葉系幹細胞、体性幹細胞および胚 性幹細胞力 なる群より選択されることを特徴とする請求の範囲 1に記載の培養基材 [14] The culture substrate according to claim 1, wherein the stem cells are selected from the group consisting of neural stem cells, hematopoietic stem cells, mesenchymal stem cells, somatic stem cells, and embryonic stem cells.
[15] 幹細胞を分化させるための培養基材。 [15] A culture substrate for differentiating stem cells.
[16] 0. 01〜： LOO /z mの範囲の膜厚を有している薄膜を備えていることを特徴とする請 求の範囲 15に記載の培養基材。  [16] 0.01-: The culture substrate according to claim 15, comprising a thin film having a film thickness in the range of LOO / zm.
[17] 前記薄膜を複数積層して備えていることを特徴とする請求の範囲 16に記載の培養 基材。 [17] The culture substrate according to claim 16, comprising a plurality of the thin films stacked.
[18] 前記薄膜が榭脂からなることを特徴とする請求の範囲 16に記載の培養基材。  [18] The culture substrate according to claim 16, wherein the thin film is made of sallow.
[19] 前記樹脂が生分解性ポリマーを含んでいることを特徴とする請求の範囲 18に記載 の培養基材。 [19] The culture substrate according to claim 18, wherein the resin contains a biodegradable polymer.
[20] 前記樹脂が両親媒性ポリマーをさらに含んでいることを特徴とする請求の範囲 19 に記載の培養基材。  [20] The culture substrate according to claim 19, wherein the resin further contains an amphiphilic polymer.
[21] 前記樹脂が生分解性ポリマーおよび両親媒性ポリマーからなることを特徴とする請 求の範囲 18に記載の培養基材。  [21] The culture substrate according to claim 18, wherein the resin comprises a biodegradable polymer and an amphiphilic polymer.
[22] 前記生分解性ポリマー力 ポリ乳酸、ポリ（ ε一力プロラタトン）、およびポリ（グリコー ル酸ー乳酸)共重合体力 なる群より選択されることを特徴とする請求の範囲 19また は 21に記載の培養基材。 [22] The biodegradable polymer strength selected from the group consisting of polylactic acid, poly (ε-strength prolatatone), and poly (glycolic acid-lactic acid) copolymer strength. The culture substrate according to 1.
[23] 前記両親媒性ポリマーが、疎水性側鎖としてドデシル基を有し親水性側鎖としてラ クトース基またはカルボキシル基を有して 、る、アクリルアミドポリマーを主鎖骨格とす る両親媒性榭脂;ポリエチレングリコール系共重合体;および、ァ-オン性高分子と長 鎖アルキルアンモ-ゥム塩とのポリイオンコンプレックス力 なる群より選択されること を特徴とする請求の範囲 20または 21に記載の培養基材。 [23] The amphiphilic polymer having a dodecyl group as a hydrophobic side chain and a lactose group or a carboxyl group as a hydrophilic side chain, and having an acrylamide polymer as a main chain skeleton. Claim 20 or 21 characterized in that it is selected from the group consisting of a resin; a polyethylene glycol copolymer; and a polyion complex force of a terpolymer and a long-chain alkyl ammonium salt. The culture substrate as described.
[24] 複数の孔を有して 、ることを特徴とする請求の範囲 15に記載の培養基材。 [24] The culture substrate according to claim 15, which has a plurality of holes.
[25] 前記複数の孔がハ-カム様に配列されていることを特徴とする請求の範囲 24に記 載の培養基材。 [25] The culture substrate according to claim 24, wherein the plurality of holes are arranged in a Herkam-like manner.
[26] 各孔が貫通して!/、ることを特徴とする請求の範囲 24に記載の培養基材。  [26] The culture substrate according to claim 24, wherein each hole penetrates! /.
[27] 各孔が連通して 、ることを特徴とする請求の範囲 24に記載の培養基材。 [27] The culture substrate according to claim 24, wherein each hole is in communication.
[28] 前記幹細胞が、神経幹細胞、造血幹細胞、間葉系幹細胞、体性幹細胞および胚 性幹細胞力もなる群より選択されることを特徴とする請求の範囲 15に記載の培養基 [28] The culture medium according to claim 15, wherein the stem cells are selected from the group consisting of neural stem cells, hematopoietic stem cells, mesenchymal stem cells, somatic stem cells, and embryonic stem cell forces.
606C0C/900Zdf/X3d LZ ム0 60/900Z OAV 606C0C / 900Zdf / X3d LZ Mu 0 60 / 900Z OAV
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