WO2017111160A1 - Cell-activating composite material, method for manufacturing cell-activating composite material, and cell culturing kit - Google Patents

Cell-activating composite material, method for manufacturing cell-activating composite material, and cell culturing kit Download PDF

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Publication number
WO2017111160A1
WO2017111160A1 PCT/JP2016/088700 JP2016088700W WO2017111160A1 WO 2017111160 A1 WO2017111160 A1 WO 2017111160A1 JP 2016088700 W JP2016088700 W JP 2016088700W WO 2017111160 A1 WO2017111160 A1 WO 2017111160A1
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composite material
cell
cells
cell activation
culture
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PCT/JP2016/088700
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French (fr)
Japanese (ja)
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土屋　利江
惠珍 朴
伊藤　博
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大日精化工業株式会社
土屋　利江
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Priority to JP2017558327A priority Critical patent/JP6680802B2/en
Publication of WO2017111160A1 publication Critical patent/WO2017111160A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres

Definitions

  • the present invention relates to a composite material for cell activation, a method for producing a composite material for cell activation, a cell culture kit, and use of the composite material for cell activation.
  • Patent Document 1 In growth culture and differentiation induction culture, for example, a factor such as cytokine is added to the cultured cells in order to promote the proliferation and differentiation induction of cultured animal cells (Patent Document 1).
  • a medium capable of maintaining the immunosuppressive function of transplanted mesenchymal stem cells is known (Patent Document 2).
  • animal cells are cultured while promoting proliferation in the presence of an extracellular matrix derived from mesenchymal cells, epithelial cells, cartilage precursor cells, or fibroblasts (Patent Document 3).
  • Patent Document 4 Specific immune rejection can be exempted by transplanting a dried product excellent in biocompatibility obtained by drying the produced extracellular matrix
  • Patent Document 5 an implant composed of chondrocytes and an extracellular matrix for treating sports disorders, osteoarthritis and the like is known (Patent Document 5).
  • Patent Document 6 a porous support for well growing and surviving cells that are uniformly and stably held and engrafted is known (Patent Document 6). Furthermore, a method for activating cells for transplantation in an electrostatic field atmosphere (Patent Document 7), a method for increasing only the production of extracellular matrix without growing cells (Patent Document 8), etc. are known. It has been.
  • the present invention has been made in view of such problems of the prior art, and the problem is to effectively activate cultured cells and cells of damaged tissues both in vivo and in vitro. It is an object of the present invention to provide a cell activation composite material that can be easily handled and can promote proliferation and differentiation induction. Moreover, the place made into the subject of this invention is providing the manufacturing method of said composite material for cell activation. Furthermore, the subject of the present invention is to provide a cell culture kit using the above-mentioned cell activation composite material, and the use of the above-mentioned cell activation composite material for promoting the growth of cultured cells and culturing them. There is to do.
  • the present inventors have lyophilized a composite material precursor obtained by supporting cells inside a porous support made of a biocompatible material. As a result, the present invention has been completed.
  • the following composite material for cell activation is provided.
  • a cell activation composite material obtained by freeze-drying a composite material precursor in which cells are supported inside a porous support made of a biocompatible material.
  • the composite material for cell activation according to [1] including the support and freeze-dried cells that are lyophilized from the cells and supported in the pores of the support.
  • the support is a sponge-like support.
  • a freeze-dried cell mass in which five or more spherical freeze-dried cells are collected is observed [2] to [2]
  • the manufacturing method of the composite material for cell activation shown below is provided.
  • a method for producing a composite material for cell activation comprising: a step (1) for obtaining a composite material precursor; and a step (2) for freeze-drying the obtained composite material precursor.
  • the following cell culture kit is provided.
  • a cell culture kit comprising the composite material for cell activation according to any one of [1] to [8].
  • a culture container is further provided, and the culture container has a culture cell storage unit that stores the culture cell, and the cell at a position that directly or indirectly contacts the culture cell stored in the culture cell storage unit.
  • the composite material for cell activation according to the present invention is capable of effectively activating cultured cells and cells of damaged tissues and promoting their proliferation and differentiation induction both in vivo and in vitro. It is easy. Further, according to the method for producing a composite material for cell activation of the present invention, the composite material for cell activation can be produced. Furthermore, according to the present invention, it is possible to provide a cell culture kit using the above-mentioned cell activation composite material, and use of the above-mentioned cell activation composite material for cultivating cells while promoting the growth of cultured cells. it can.
  • FIG. 3 is an electron micrograph showing the microstructure of the cell activation composite material of Example 1.
  • FIG. 3 is an electron micrograph showing the microstructure of the cell activation composite material of Example 2.
  • FIG. 4 is an electron micrograph showing the microstructure of the cell activation composite material of Example 3.
  • composite material for cell activation of the present invention (hereinafter also simply referred to as “composite material”) is obtained by lyophilizing a composite material precursor in which cells are supported inside a porous support made of a biocompatible material. It is obtained. The details will be described below.
  • factors that activate live cells and promote their proliferation and differentiation are intracellular. It is included as an ingredient. And it is thought that the intracellular component (various factors) which flowed out from the cell whose cell membrane was broken by freeze-drying is supported in the pores of the support existing around. For this reason, when the composite material of the present invention is arranged so as to come into contact with cultured cells or damaged tissues, various factors carried in the pores are relatively quickly released from the support at the beginning of the culture start or the healing start. Released.
  • the composite material of the present invention is such that intracellular components including freeze-dried cells and various factors are carried inside the porous support (more specifically, in the pores), the support It is easier to handle than lyophilized cells themselves that do not have Furthermore, since the cell activation is not inhibited by the support, it is possible to effectively activate cultured cells and cells of damaged tissues to promote their proliferation and differentiation induction.
  • the porous support constituting the composite material of the present invention is formed of a biocompatible material.
  • the biocompatible material may be biodegradable or non-biodegradable.
  • the biocompatible material may be a natural product or a synthetic product. These materials may be chemically modified with a functional group such as a sulfate group.
  • biocompatible materials include proteins such as gelatin, collagen, elastin, fibroin and albumin; polysaccharides such as hyaluronic acid, cellulose, chitin, chitosan, starch, pectin, alginic acid, agarose and agar And derivatives thereof. Moreover, it is also possible to use the commercial item which used these natural products as a raw material.
  • biocompatible materials specific examples of synthetic materials include peptides, polylactic acid, polyglycolic acid, poly- ⁇ -caprolactone and other polymers; copolymers of monomers constituting these polymers; ceramics and the like Can be mentioned. These biocompatible materials can be used singly or in combination of two or more.
  • biodegradable materials are preferably subjected to insolubilization treatment such as crosslinking.
  • insolubilization treatment such as crosslinking.
  • biodegradable materials such as gelatin, collagen, and synthetic peptides because of their adhesiveness to tissues.
  • synthetic peptide a synthetic peptide synthesized organically, a synthetic peptide produced by genetic engineering, or the like can be used. More specifically, in addition to artificial collagen and artificial gelatin imitating natural peptides, peptides having novel physical properties can be used.
  • a large number of pores for holding freeze-dried cells are formed inside the support.
  • As the support in addition to a sponge-like support, non-woven fabric, woven fabric, or the like can be used.
  • the pore diameter is preferably 5 to 1000 ⁇ m, more preferably 10 to 500 ⁇ m.
  • a cell suspension obtained by suspending cells in a liquid medium that has been brought into contact with a support to produce the composite material of the present invention enters the pores. It's easy to do.
  • the cells are unlikely to flow out of the pores, but the liquid medium is preferable because it flows out easily.
  • the composite material of the present invention is used for effectively activating cultured cells and damaged tissue cells to promote their proliferation and differentiation induction.
  • the shape maintaining period of the support is preferably a period until cell proliferation and differentiation induction are completed (in vivo, a period until treatment and repair of the affected area are completed).
  • the shape maintaining period of the support can be adjusted by introducing intermolecular crosslinks into the biodegradable material.
  • the biodegradable material is a protein
  • it can be crosslinked by chemical crosslinking, physical crosslinking, or crosslinking using an enzyme.
  • a chemical crosslinking agent such as formalin, glutaraldehyde, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether, glycerol polyglycidyl ether, hexamethylene diisocyanate can be used.
  • biodegradable material For physical crosslinking, ⁇ rays, ultraviolet rays, or the like can be used. In addition, an enzyme such as transglutaminase can be used for crosslinking using an enzyme.
  • a dehydrating agent such as carbodiimide, a Ca salt, or the like in addition to the chemical crosslinking agent.
  • the size and shape of the support are not particularly limited, and may be a size and a shape corresponding to the application.
  • Specific examples of the shape of the support include a cube, a cylinder, and a sheet.
  • Freeze-dried cells are formed by freeze-drying the cells carried inside the porous support.
  • Examples of cells include non-human embryonic stem cell lines, human embryonic stem cell lines, induced pluripotent stem cells, spermatogonial stem cells, embryonic stem cells, mesenchymal stem cells, endothelial stem cells, hematopoietic stem cells, neural stem cells, neural crest stem cells, and gastrointestinal stem cells.
  • Stem cells such as bone marrow, adipose tissue, placental tissue, umbilical cord tissue, umbilical cord blood, and dental pulp; human dermal fibroblasts, endothelial cells, gastrointestinal epithelial cells, mammary epithelial cells, melanocytes , Lung epithelial cells, renal epithelial cells, keratinocytes, urothelial cells, hepatocytes, corneal cells, lens cells, osteoblasts, bone cells, odontoblasts, chondrocytes, ligament cells, tendon cells, pituitary gland Cells, salivary gland cells, adrenal cells, exocrine pancreatic cells, endocrine pancreatic cells, Leydig cells, adipocytes, fibroblasts, retinal cells, dopaminergic neurons, GABAergic neurons, glutamatergic Uron, cholinergic neuron, adrenergic neuron, astrocyte, oligodendrocyte, Schwann cell, smooth
  • components other than freeze-dried cells and intracellular components may be further supported in the pores of the support.
  • other components include a medium-containing component, hyaluronic acid, a chemically modified product of hyaluronic acid, iron sulfate (FeSO 4 ), chondroitin sulfate, and various cytokines.
  • the porous support constituting the composite material of the present invention is manufactured using a raw material collected from a breeding-managed animal such as prion or a contamination-free synthetic material. Is preferred.
  • the composite material of the present invention is preferably substantially free of viable bacteria. By substantially not containing viable bacteria, it can be suitably used for culturing cultured cells and transplanting to a site in a living body (such as a human or non-human animal) in which cells or tissues are defective. .
  • substantially free of viable bacteria means that microorganisms (bacteria or fungi, etc.) are detected when tested according to the “sterility test method” defined in the Japanese Pharmacopoeia General Test Method. Means not.
  • the composite material may be produced by an aseptic operation.
  • a composite material is transplanted into a site in a living body (such as a human or a non-human animal) in which cells or tissues are defective.
  • a living body such as a human or a non-human animal
  • various factors released from the composite material can be effectively acted on surrounding cells, and cell proliferation and differentiation induction can be promoted to promote healing.
  • the composite material of the present invention utilizes various factors that promote cell proliferation and differentiation induction, not the cultured cells themselves. For this reason, the composite material of the present invention is not subject to so-called regenerative medicine regulation and can be easily spread widely.
  • the method for producing a composite material for cell activation of the present invention includes a step of obtaining a composite material precursor by supporting cells in pores of a porous support made of a biocompatible material (step (1)), A step of freeze-drying the obtained composite material precursor (step (2)). The details will be described below.
  • step (1) cells are supported in the pores of the porous support described above to obtain a composite material precursor.
  • a cell suspension in which cells are suspended in a liquid medium is brought into contact with the support, and the cell suspension enters (soaks) into the pores.
  • the number of cells in the composite material precursor is preferably 10 4 or more, and more preferably 10 7 or more, per apparent volume of 125 mm 3 of the support.
  • step (2) the composite material precursor obtained in step (1) is freeze-dried.
  • the composite material for cell activation of this invention can be obtained.
  • the predetermined time is preferably a short time, and is preferably within 60 minutes. If it exceeds 60 minutes, undesirable effects may appear on the cells.
  • the composite material precursor can be freeze-dried without using a water-soluble freeze-drying protective agent such as dimethyl sulfoxide (DMSO) or glycerin that is generally used for freeze-drying cells.
  • DMSO dimethyl sulfoxide
  • the composite material precursor is preferably frozen at ⁇ 50 ° C. or lower, and more preferably at ⁇ 80 ° C. or lower.
  • Freeze-drying can be performed using, for example, a general freezer or a cryogenic freezer such as a deep freezer.
  • the composite material precursor is frozen using a cooling medium (liquefied gas) such as carbon dioxide ( ⁇ 78.5 ° C.), liquid nitrogen ( ⁇ 196 ° C.), liquid helium ( ⁇ 269 ° C.), and then dried.
  • a cooling medium liquefied gas
  • lyophilization using an inert gas liquefied gas such as nitrogen or helium is preferable because it has little influence on cells and high safety.
  • it may be frozen by lowering the temperature step by step, or it may be frozen by lowering the temperature at once, but freezing the precursor of the composite material by lowering the temperature at once promotes proliferation and differentiation induction.
  • the composite material superior in ability to be obtained can be obtained. Specifically, since the ratio of frozen cells whose original shape is maintained without breaking the cell membrane can be increased, the composite material has a higher ability to promote proliferation and differentiation induction. After freezing, it is preferable to immediately sublimate water under reduced pressure and dry, as long as the composite material precursor is frozen during drying by sublimation. It may be dried using a freeze dryer, or may be quickly put into a sealed container after freezing and dried under reduced pressure.
  • the cell culture kit of the present invention comprises the above-described composite material for cell activation of the present invention.
  • the above-described composite material can effectively act on cultured cells and activate the cultured cells to promote proliferation. For this reason, by using this composite material, it is possible to construct a cell culture kit capable of culturing various cultured cells that have been slow to grow and difficult to culture.
  • the configuration of the cell culture kit of the present invention can be the same as that of a general conventional cell culture kit except that the above-described composite material is provided.
  • the size (diameter) of a well, the number of wells per one cell culture plate, etc. are not specifically limited.
  • the diameter of the well is, for example, 0.64 to 35 mm.
  • the number of wells per cell culture plate is, for example, 4 to 96.
  • a petri dish as shown in FIG. 2 can also be used as the culture container 20. That is, a cell culture kit can be obtained by housing the composite material in the culture vessel 20.
  • the size (diameter) of the petri dish is not particularly limited. The diameter of the petri dish is, for example, 35 to 150 mm. Commercially available cell culture plates and petri dishes can be used.
  • the cultured cells and the medium are accommodated in the well 5 and a composite material is disposed so as to be in contact with the accommodated cultured cells and medium. That's fine.
  • the cultured cells and the medium are accommodated in the culture container 20 and the composite material is brought into contact with the accommodated cultured cells and medium. What is necessary is just to arrange.
  • FIG. 3 is a schematic view showing one embodiment of the cell culture kit of the present invention.
  • the cell culture kit 100 of the embodiment shown in FIG. 3 includes a culture container 50 having a cultured cell storage unit 35 and a composite material storage unit 45.
  • the cultured cell storage unit 35 is a part (space) that stores the cultured cells 31 and the culture medium 33.
  • the composite material housing 45 is a portion (space) in which the composite material 43 is disposed.
  • the composite material 43 accommodated in the composite material accommodating portion 45 indirectly with the cultured cells 31 accommodated in the cultured cell accommodating portion 35 with the diaphragm 47 constituting the bottom of the composite material accommodating portion 45 interposed therebetween. Contact.
  • the diaphragm 47 is a film in which various components and medium components flowing out from the composite material 43 are permeated, but the placed composite material 43 is formed with a hole of a size that does not fall downward. With the configuration as shown in FIG. 3, various factors released from the composite material can be cultured while acting on cultured cells without applying an excessive load, and promoting proliferation and differentiation induction.
  • the size of the composite material to be used may be appropriately set according to the size of the well or petri dish of the cell culture plate.
  • the composite material may be manufactured in advance to have a desired size in consideration of the size of a well or the like, or may be cut to have a desired size.
  • Examples of cells (cultured cells) that can be cultured using the cell culture kit of the present invention include the same cells as those used to form the above-mentioned freeze-dried cells. Among these, it is particularly effective when culturing slow-growing cells such as mesenchymal stem cells and primary cells.
  • Example 1 The cross-linked gelatin sponge was aseptically cut into a cylindrical shape having a diameter of 6 mm and a height of 5 mm. A suspension of V79 cells (fibroblasts) (10 6 cells / 50 ⁇ L, MEM medium + 2 mmol / L glutamine) was placed on a cylindrical gelatin sponge and allowed to stand for 30 minutes. So that a composite material precursor was obtained. In addition, as V79 cell, what was obtained from JCRB cell bank (cell registration number JCRB0603, Lot No. 10012012) was used. After instantly freezing the composite material precursor using liquid nitrogen, it was transferred to a lyophilizer (trade name “FD-5N”, manufactured by EYELA Co., Ltd.) and immediately evacuated and lyophilized to obtain a composite material. .
  • a lyophilizer trade name “FD-5N”, manufactured by EYELA Co., Ltd.
  • a part of the obtained composite material was cut aseptically to prepare two sample pieces for sterility test in a cylindrical shape (diameter 6 mm, height 1 mm).
  • a sterility test was performed according to the “sterility test method” defined in the Japanese Pharmacopoeia General Test Method. Specifically, the prepared sample pieces were placed in a liquid thioglycolic acid medium and a soybean / casein / digest medium, respectively, and cultured for 14 days. As a result, since no growth of microorganisms was observed in any of the media, the composite material was judged to be substantially sterile.
  • the sterility test was performed on all manufactured composite materials in the same manner as described above, and it was confirmed that all the composite materials were substantially sterile.
  • Example 2 The composite material precursor obtained in Example 1 was frozen in a deep freezer (product number “CLN-30UW”, manufactured by Nippon Freezer Co., Ltd.) at ⁇ 80 ° C. and then freeze-dried in the same manner as in Example 1 to form a composite. Obtained material.
  • Example 3 The composite material precursor obtained in Example 1 was placed in a freezer (product number “MDF-MU300H-P”, manufactured by Panasonic Hercare) at ⁇ 20 ° C., and further frozen in stages using liquid nitrogen. . Subsequently, it lyophilized similarly to Example 1 and the composite material was obtained.
  • Comparative Example 1 The cylindrical gelatin sponge (without lyophilized cells) used in Example 1 was used as the material of Comparative Example 1.
  • Example 4 A composite material was obtained in the same manner as in Example 1 except that a suspension of V79 cells (fibroblasts) (10 5 cells / 50 ⁇ L, MEM medium + 2 mmol / L glutamine) was used.
  • Example 5 The cross-linked gelatin sponge was aseptically cut into a cylindrical shape having a diameter of 6 mm and a height of 5 mm.
  • a suspension of hMSC cells human mesenchymal stem cells (10 6 cells / 50 ⁇ L, MEM medium + 2 mmol / L glutamine) was placed on a cylindrical gelatin sponge and allowed to stand for 30 minutes.
  • a composite material precursor was obtained by soaking into a gelatin sponge.
  • hMSC cells those obtained from Lonza were used.
  • the composite material precursor was instantly frozen using liquid nitrogen, then transferred to a lyophilizer, and immediately evacuated and vacuum-dried to obtain a composite material.
  • Comparative Example 2 The cylindrical gelatin sponge (without lyophilized cells) used in Example 5 was used as the material of Comparative Example 2.
  • Example 6 A composite material was obtained in the same manner as in Example 5 except that a suspension of hMSC cells (human mesenchymal stem cells) (10 5 cells / 50 ⁇ L, MEM medium + 2 mmol / L glutamine) was used.
  • hMSC cells human mesenchymal stem cells
  • hMSC cells 4 ⁇ 10 3 cells were suspended in 2 mL of liquid medium (Lonza, MSCGM Bulletkit). The obtained suspension was placed in each well of four cell culture plates (24 wells) and cultured for 18 hours in a carbon dioxide incubator. After culturing, the composite material of Example 5 is put into a culture insert (pore size: 8 ⁇ m, manufactured by Falcon) having a hole that does not drop the composite material (Evaluation Example 11), and the material of Comparative Example 2 is used. The plate was divided into four plates (Evaluation Example 12), the composite material of Example 6 (Evaluation Example 13), and nothing (Evaluation Example 14), and cultured in a carbon dioxide incubator for 6 days.
  • the number of cells was counted using a reagent for measuring the number of living cells (trade name “WST-8” (Cell Count reagent SF), manufactured by Hanai Chemical Co., Ltd.), and the average value per well was calculated. The results are shown in Table 4.
  • the composite material for cell activation of the present invention is useful as a material for effectively activating cultured cells and cells of damaged tissues and promoting their proliferation and differentiation induction.

Abstract

Provided is an easy-to-handle cell-activating composite material that is capable of effectively activating, both in vivo and in vitro, cultured cells or cells from damaged tissue and promoting the proliferation or differentiation thereof. The cell-activating composite material is obtained by lyophilizing a composite material precursor, in which cells are supported within a porous supporting body comprising a biocompatible material, such as gelatin or collagen. The cell-activating composite material comprises said supporting body as well as lyophilized cells, which are cells that are lyophilized and supported within the pores of the supporting body. The biocompatible material is a bioabsorbable material, such as gelatin or collagen.

Description

細胞活性化用複合材料、細胞活性化用複合材料の製造方法、及び細胞培養キットComposite material for cell activation, method for producing composite material for cell activation, and cell culture kit
　本発明は、細胞活性化用複合材料、細胞活性化用複合材料の製造方法、細胞培養キット、及び細胞活性化用複合材料の使用に関する。 The present invention relates to a composite material for cell activation, a method for producing a composite material for cell activation, a cell culture kit, and use of the composite material for cell activation.
　再生医療やｉｎ　ｖｉｔｒｏで産生した細胞外基質の利用など、細胞の活性を利用した治療などが盛んに行われている。このため、細胞の活性を高め、細胞外基質の産生量を向上させる技術について広く検討されている。そのうち、幹細胞を含めた各種動物細胞等の増殖培養及び分化誘導培養において、培養細胞の増殖や分化誘導を促進するとともに、動物幹細胞等の分化能を増殖中に効果的に維持し、かつ、免疫反応を抑制し、従来の培養方法と比較して多くの動物細胞を得ることが可能な培養方法が知られている。さらに、積極的に産生させた細胞外基質を治療部位へ用いるための移植物なども知られている。 Regenerative medicine and treatment using cell activity such as the use of extracellular matrix produced in vitro are actively performed. For this reason, techniques for increasing the activity of cells and improving the production of extracellular matrix have been widely studied. Among them, in the proliferation culture and differentiation induction culture of various animal cells including stem cells, the proliferation and differentiation induction of the cultured cells are promoted, the differentiation ability of animal stem cells and the like is effectively maintained during the proliferation, and A culture method that suppresses the reaction and can obtain more animal cells than conventional culture methods is known. Furthermore, an implant for using an extracellular matrix actively produced at a treatment site is also known.
　増殖培養及び分化誘導培養において、培養動物細胞の増殖や分化誘導を促進すべく、例えば、培養細胞にサイトカイン等の因子を添加することが行われている（特許文献１）。また、移植する間葉系幹細胞の免疫抑制機能を維持可能な培地が知られている（特許文献２）。さらに、間葉系細胞、上皮系細胞、軟骨前駆細胞、又は線維芽細胞由来の細胞外基質の存在下において、増殖を促進しながら動物細胞を培養することが知られている（特許文献３）。また、産生した細胞外基質を乾燥して得られる、生体適合性に優れた乾燥物を移植することで、特異的な免疫拒絶を免除できることが知られている（特許文献４）。 In growth culture and differentiation induction culture, for example, a factor such as cytokine is added to the cultured cells in order to promote the proliferation and differentiation induction of cultured animal cells (Patent Document 1). In addition, a medium capable of maintaining the immunosuppressive function of transplanted mesenchymal stem cells is known (Patent Document 2). Furthermore, it is known that animal cells are cultured while promoting proliferation in the presence of an extracellular matrix derived from mesenchymal cells, epithelial cells, cartilage precursor cells, or fibroblasts (Patent Document 3). . In addition, it is known that specific immune rejection can be exempted by transplanting a dried product excellent in biocompatibility obtained by drying the produced extracellular matrix (Patent Document 4).
　一方、事故等によって損傷した組織や不全となった臓器を治療すべく、種々の生体組織再生に用いられる移植物などが研究されている。例えば、スポーツ障害や変形性関節症等を治療するための、軟骨細胞と細胞外基質で構成される移植物が知られている（特許文献５）。 On the other hand, in order to treat a tissue damaged by an accident or an organ that has failed, various implants used for regeneration of living tissue have been studied. For example, an implant composed of chondrocytes and an extracellular matrix for treating sports disorders, osteoarthritis and the like is known (Patent Document 5).
　また、均一かつ安定して保持及び生着させた細胞を良好に増殖及び生存させるための多孔性支持体が知られている（特許文献６）。さらに、移植用の細胞を静電場雰囲気内に置いて活性化する方法（特許文献７）や、細胞を増殖させることなく、細胞外マトリックスの産生のみを増大させる方法（特許文献８）などが知られている。 In addition, a porous support for well growing and surviving cells that are uniformly and stably held and engrafted is known (Patent Document 6). Furthermore, a method for activating cells for transplantation in an electrostatic field atmosphere (Patent Document 7), a method for increasing only the production of extracellular matrix without growing cells (Patent Document 8), etc. are known. It has been.
特表２０１１－５１６４３６号公報Special table 2011-516436 gazette 国際公開第２０１１／１１１７８７号International Publication No. 2011/111787 特開２００６－００００５９号公報JP 2006-000059 A 特表２０１０－５０４０９３号公報Japanese translation of PCT publication 2010-504093 国際公開第２００４／０５２４１８号International Publication No. 2004/052418 特開２００４－２１６１１９号公報JP 2004-216119 A 国際公開第２００６／０８５５３４号International Publication No. 2006/085534 特表２００４－５０７２３７号公報JP-T-2004-507237
　しかし、従来の技術においては、厳密に条件を整えた培養操作が必要であるとともに、使用する際にその都度細胞を培養して製造する必要があるため、予め製造しておいたものを保管しておくことはできなかった。また、ｉｎ　ｖｉｖｏ及びｉｎ　ｖｉｔｒｏのいずれにおいても培養細胞や損傷組織の細胞を活性化させうる材料は、これまで存在しなかった。 However, in the conventional technique, it is necessary to carry out culturing operations with strictly conditioned conditions, and it is necessary to culture and produce cells each time they are used. I couldn't keep it. In addition, there has been no material that can activate cultured cells and cells of damaged tissues both in vivo and in vitro.
　本発明は、このような従来技術の有する問題点に鑑みてなされたものであり、その課題とするところは、ｉｎ　ｖｉｖｏ及びｉｎ　ｖｉｔｒｏのいずれにおいても、培養細胞や損傷組織の細胞を有効に活性化させてその増殖や分化誘導を促進させることが可能な、取り扱いが容易な細胞活性化用複合材料を提供することにある。また、本発明の課題とするところは、上記の細胞活性化用複合材料の製造方法を提供することにある。さらに、本発明の課題とするところは、上記の細胞活性化用複合材料を用いた細胞培養キット、及び培養細胞の増殖を促進させて培養するための上記細胞活性化用複合材料の使用を提供することにある。 The present invention has been made in view of such problems of the prior art, and the problem is to effectively activate cultured cells and cells of damaged tissues both in vivo and in vitro. It is an object of the present invention to provide a cell activation composite material that can be easily handled and can promote proliferation and differentiation induction. Moreover, the place made into the subject of this invention is providing the manufacturing method of said composite material for cell activation. Furthermore, the subject of the present invention is to provide a cell culture kit using the above-mentioned cell activation composite material, and the use of the above-mentioned cell activation composite material for promoting the growth of cultured cells and culturing them. There is to do.
　本発明者らは上記課題を解決すべく鋭意検討した結果、生体親和性材料からなる多孔性の支持体の内部に細胞を担持させて得た複合材料前駆体を凍結乾燥することによって、上記課題を解決することが可能であることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have lyophilized a composite material precursor obtained by supporting cells inside a porous support made of a biocompatible material. As a result, the present invention has been completed.
　すなわち、本発明によれば、以下に示す細胞活性化用複合材料が提供される。
　［１］生体親和性材料からなる多孔性の支持体の内部に細胞を担持させた複合材料前駆体を凍結乾燥して得られる細胞活性化用複合材料。
　［２］前記支持体と、前記支持体の細孔内に担持された、前記細胞が凍結乾燥した凍結乾燥細胞と、を含む前記［１］に記載の細胞活性化用複合材料。
　［３］前記支持体の前記細孔内に担持された、培地含有成分、ヒアルロン酸、ヒアルロン酸の化学修飾物、硫酸鉄（ＦｅＳＯ4）、コンドロイチン硫酸、及びサイトカインからなる群より選択される少なくとも一種をさらに含む前記［２］に記載の細胞活性化用複合材料。
　［４］前記生体親和性材料が、ゼラチン、コラーゲン、及び合成ペプチドの少なくともいずれかの生体内分解性材料である前記［１］～［３］のいずれかに記載の細胞活性化用複合材料。
　［５］前記支持体がスポンジ状支持体である前記［１］～［４］のいずれかに記載の細胞活性化用複合材料。
　［６］前記細胞が、間葉系幹細胞、体細胞、遺伝子組み換え細胞、癌細胞株、及び不死化細胞株からなる群より選択される少なくとも一種である前記［１］～［５］のいずれかに記載の細胞活性化複合材料。
　［７］生菌を実質的に含まない前記［１］～［６］のいずれかに記載の細胞活性化用複合材料。
　［８］内部の任意の領域を走査型電子顕微鏡により５００倍に拡大して観察した場合に、球状の前記凍結乾燥細胞が５個以上集まった凍結乾燥細胞塊が観察される前記［２］～［７］のいずれかに記載の細胞活性化用複合材料。 That is, according to the present invention, the following composite material for cell activation is provided.
[1] A cell activation composite material obtained by freeze-drying a composite material precursor in which cells are supported inside a porous support made of a biocompatible material.
[2] The composite material for cell activation according to [1], including the support and freeze-dried cells that are lyophilized from the cells and supported in the pores of the support.
[3] At least selected from the group consisting of a medium-containing component, hyaluronic acid, a chemically modified product of hyaluronic acid, iron sulfate (FeSO 4 ), chondroitin sulfate, and cytokine supported in the pores of the support The composite material for cell activation according to the above [2], further comprising one kind.
[4] The cell activation composite material according to any one of [1] to [3], wherein the biocompatible material is a biodegradable material of at least one of gelatin, collagen, and synthetic peptides.
[5] The cell activation composite material according to any one of [1] to [4], wherein the support is a sponge-like support.
[6] Any one of [1] to [5], wherein the cell is at least one selected from the group consisting of mesenchymal stem cells, somatic cells, genetically modified cells, cancer cell lines, and immortalized cell lines. A cell-activated composite material according to 1.
[7] The cell activation composite material according to any one of [1] to [6], which is substantially free from viable bacteria.
[8] When an arbitrary region inside is observed with a scanning electron microscope at a magnification of 500 times, a freeze-dried cell mass in which five or more spherical freeze-dried cells are collected is observed [2] to [2] [7] The composite material for cell activation according to any one of [7].
　また、本発明によれば、以下に示す細胞活性化用複合材料の製造方法が提供される。
　［９］前記［１］～［８］のいずれかに記載の細胞活性化用複合材料の製造方法であって、生体親和性材料からなる多孔性の支持体の細孔内に細胞を担持させて複合材料前駆体を得る工程（１）と、得られた前記複合材料前駆体を凍結乾燥する工程（２）と、を有する細胞活性化用複合材料の製造方法。
　［１０］前記工程（２）における、前記複合材料前駆体を凍結する温度が－５０℃以下である前記［９］に記載の細胞活性化用複合材料の製造方法。 Moreover, according to this invention, the manufacturing method of the composite material for cell activation shown below is provided.
[9] The method for producing a cell activation composite material according to any one of [1] to [8], wherein cells are supported in the pores of a porous support made of a biocompatible material. A method for producing a composite material for cell activation, comprising: a step (1) for obtaining a composite material precursor; and a step (2) for freeze-drying the obtained composite material precursor.
[10] The method for producing a composite material for cell activation according to the above [9], wherein the temperature for freezing the composite material precursor in the step (2) is −50 ° C. or lower.
　さらに、本発明によれば、以下に示す細胞培養キットが提供される。
　［１１］前記［１］～［８］のいずれかに記載の細胞活性化用複合材料を備えた細胞培養キット。
　［１２］培養容器をさらに備え、前記培養容器は、培養細胞を収容する培養細胞収容部と、前記培養細胞収容部内に収容された前記培養細胞と直接的又は間接的に接触する位置に前記細胞活性化用複合材料を配置する複合材料収容部と、を有する前記［１１］に記載の細胞培養キット。 Furthermore, according to the present invention, the following cell culture kit is provided.
[11] A cell culture kit comprising the composite material for cell activation according to any one of [1] to [8].
[12] A culture container is further provided, and the culture container has a culture cell storage unit that stores the culture cell, and the cell at a position that directly or indirectly contacts the culture cell stored in the culture cell storage unit. The cell culture kit according to [11], further including a composite material container in which the composite material for activation is arranged.
　また、本発明によれば、以下に示す細胞活性化用複合材料の使用が提供される。
　［１３］培養細胞を培養するための、前記［１］～［８］のいずれかに記載の細胞活性化用複合材料の使用。 Moreover, according to this invention, use of the composite material for cell activation shown below is provided.
[13] Use of the composite material for cell activation according to any one of [1] to [8] for culturing cultured cells.
　本発明の細胞活性化用複合材料は、ｉｎ　ｖｉｖｏ及びｉｎ　ｖｉｔｒｏのいずれにおいても、培養細胞や損傷組織の細胞を有効に活性化させてその増殖や分化誘導を促進させることが可能な、取り扱いが容易なものである。また、本発明の細胞活性化用複合材料の製造方法によれば、上記の細胞活性化用複合材料を製造することができる。さらに、本発明によれば、上記の細胞活性化用複合材料を用いた細胞培養キット、及び培養細胞の増殖を促進させて培養するための上記細胞活性化用複合材料の使用を提供することができる。 The composite material for cell activation according to the present invention is capable of effectively activating cultured cells and cells of damaged tissues and promoting their proliferation and differentiation induction both in vivo and in vitro. It is easy. Further, according to the method for producing a composite material for cell activation of the present invention, the composite material for cell activation can be produced. Furthermore, according to the present invention, it is possible to provide a cell culture kit using the above-mentioned cell activation composite material, and use of the above-mentioned cell activation composite material for cultivating cells while promoting the growth of cultured cells. it can.
本発明の細胞培養キットを構成する培養容器の一例（セルカルチャープレート）を示す模式図である。It is a schematic diagram which shows an example (cell culture plate) of the culture container which comprises the cell culture kit of this invention. 本発明の細胞培養キットを構成する培養容器の他の例（シャーレ）を示す模式図である。It is a schematic diagram which shows the other example (petri dish) of the culture container which comprises the cell culture kit of this invention. 本発明の細胞培養キットの一実施形態を示す模式図である。It is a schematic diagram which shows one Embodiment of the cell culture kit of this invention. Ｖ７９細胞の培養結果を示すグラフである。It is a graph which shows the culture result of V79 cell. 実施例１の細胞活性化用複合材料の微細構造を示す電子顕微鏡写真である。3 is an electron micrograph showing the microstructure of the cell activation composite material of Example 1. FIG. 実施例２の細胞活性化用複合材料の微細構造を示す電子顕微鏡写真である。3 is an electron micrograph showing the microstructure of the cell activation composite material of Example 2. FIG. 実施例３の細胞活性化用複合材料の微細構造を示す電子顕微鏡写真である。4 is an electron micrograph showing the microstructure of the cell activation composite material of Example 3. FIG.
＜細胞活性化用複合材料＞
　以下、本発明の実施の形態について説明するが、本発明は以下の実施の形態に限定されるものではない。本発明の細胞活性化用複合材料（以下、単に「複合材料」とも記す）は、生体親和性材料からなる多孔性の支持体の内部に細胞を担持させた複合材料前駆体を凍結乾燥して得られるものである。以下、その詳細について説明する。 <Composite materials for cell activation>
Embodiments of the present invention will be described below, but the present invention is not limited to the following embodiments. The composite material for cell activation of the present invention (hereinafter also simply referred to as “composite material”) is obtained by lyophilizing a composite material precursor in which cells are supported inside a porous support made of a biocompatible material. It is obtained. The details will be described below.
　動物などの生きた細胞を、生体親和性材料からなる多孔性の支持体の細孔内に担持した状態で凍結乾燥すると、一部の細胞については細胞膜が破れることなく保持され、元の形状を維持した凍結乾燥細胞が形成される。一方、残りの細胞については、凍結乾燥により細胞膜が破れてしまい、細胞内成分が流出する。本発明の複合材料は、凍結乾燥細胞及び細胞内成分が支持体の細孔内に担持されている。より具体的には、内部の任意の領域を走査型電子顕微鏡により５００倍に拡大して観察した場合に、球状の凍結乾燥細胞が５個以上集まった凍結乾燥細胞塊が観察されるものである。 When living cells such as animals are lyophilized in a state where they are supported in the pores of a porous support made of a biocompatible material, some of the cells are retained without being broken, and the original shape is maintained. Maintained lyophilized cells are formed. On the other hand, with respect to the remaining cells, the cell membrane is broken by lyophilization, and intracellular components flow out. In the composite material of the present invention, freeze-dried cells and intracellular components are supported in the pores of the support. More specifically, a freeze-dried cell mass in which five or more spherical freeze-dried cells are gathered is observed when an arbitrary region inside is magnified 500 times with a scanning electron microscope. .
　凍結乾燥細胞の内部には、生きた細胞を活性化させてその増殖や分化誘導を促進させる因子（例えば、ＦＧＦ、ＨＧＦ、ＶＥＧＦ、ＴＩＭＰ等。以下、単に「各種因子」とも記す）が細胞内成分として含まれている。そして、凍結乾燥によって細胞膜が破れた細胞から流出した細胞内成分（各種因子）は、周囲に存在する支持体の細孔内に担持されていると考えられる。このため、本発明の複合材料を培養細胞や損傷組織と接触するように配置すると、培養開始又は治癒開始の初期には、細孔内に担持されていた各種因子が支持体から比較的速やかに放出される。そして、細胞膜が破れることなく元の形状が維持された凍結乾燥細胞からは、長期間にわたって各種因子が徐々に放出される。このため、本発明の複合材料を用いれば、培養細胞や損傷組織に長期にわたって有効に作用し、細胞を活性化させることができる。 Within the freeze-dried cells, factors that activate live cells and promote their proliferation and differentiation (eg, FGF, HGF, VEGF, TIMP, etc .; hereinafter also simply referred to as “various factors”) are intracellular. It is included as an ingredient. And it is thought that the intracellular component (various factors) which flowed out from the cell whose cell membrane was broken by freeze-drying is supported in the pores of the support existing around. For this reason, when the composite material of the present invention is arranged so as to come into contact with cultured cells or damaged tissues, various factors carried in the pores are relatively quickly released from the support at the beginning of the culture start or the healing start. Released. Various factors are gradually released over a long period of time from freeze-dried cells whose original shape is maintained without breaking the cell membrane. For this reason, if the composite material of this invention is used, it will act on a cultured cell and damaged tissue effectively over a long term, and a cell can be activated.
　また、本発明の複合材料は、多孔性の支持体の内部（より具体的には細孔内）に凍結乾燥細胞と各種因子を含む細胞内成分が担持されているものであるため、支持体を有しない凍結乾燥細胞自体と比べて取扱いが容易である。さらに、支持体によって細胞の活性化が阻害されることもないため、培養細胞や損傷組織の細胞を有効に活性化させてその増殖や分化誘導を促進させることができる。 In addition, since the composite material of the present invention is such that intracellular components including freeze-dried cells and various factors are carried inside the porous support (more specifically, in the pores), the support It is easier to handle than lyophilized cells themselves that do not have Furthermore, since the cell activation is not inhibited by the support, it is possible to effectively activate cultured cells and cells of damaged tissues to promote their proliferation and differentiation induction.
（支持体）
　本発明の複合材料を構成する多孔性の支持体は、生体親和性材料によって形成されている。生体親和性材料は、生体内分解性であっても、生体内非分解性であってもよい。また、生体親和性材料は、天然物及び合成物のいずれであってもよい。これらの材料は、硫酸基等の官能基で化学修飾されたものであってもよい。 (Support)
The porous support constituting the composite material of the present invention is formed of a biocompatible material. The biocompatible material may be biodegradable or non-biodegradable. The biocompatible material may be a natural product or a synthetic product. These materials may be chemically modified with a functional group such as a sulfate group.
　生体親和性材料のうちの天然物の具体例としては、ゼラチン、コラーゲン、エラスチン、フィブロイン、アルブミン等のタンパク質；ヒアルロン酸、セルロース、キチン、キトサン、でんぷん、ペクチン、アルギン酸、アガロース、寒天等の多糖類；及びこれらの誘導体を挙げることができる。また、これらの天然物を原料とした市販品を用いることも可能である。生体親和性材料のうち、合成物の具体例としては、ペプチド、ポリ乳酸、ポリグリコール酸、ポリ－ε－カプロラクトン等の重合体；これらの重合体を構成するモノマーの共重合体；セラミックス等を挙げることができる。これらの生体親和性材料は、１種単独で又は２種以上を組み合わせて用いることができる。これらの生体親和性材料のうち、生体内分解性材料については架橋などの不溶化処理をすることが好ましい。これらの生体親和性材料のなかでも、生体内分解性材料であるゼラチン、コラーゲン、及び合成ペプチドの少なくともいずれかを用いることが、組織へ固着性があるために好ましい。合成ペプチドとしては、有機合成される合成ペプチドや、遺伝子工学的に作製される合成ペプチドなどを用いることができる。より具体的には、天然のペプチドを模した人工コラーゲン及び人工ゼラチンの他、新規な物性を有するペプチドなどを使用することができる。 Specific examples of natural products among biocompatible materials include proteins such as gelatin, collagen, elastin, fibroin and albumin; polysaccharides such as hyaluronic acid, cellulose, chitin, chitosan, starch, pectin, alginic acid, agarose and agar And derivatives thereof. Moreover, it is also possible to use the commercial item which used these natural products as a raw material. Among biocompatible materials, specific examples of synthetic materials include peptides, polylactic acid, polyglycolic acid, poly-ε-caprolactone and other polymers; copolymers of monomers constituting these polymers; ceramics and the like Can be mentioned. These biocompatible materials can be used singly or in combination of two or more. Of these biocompatible materials, biodegradable materials are preferably subjected to insolubilization treatment such as crosslinking. Among these biocompatible materials, it is preferable to use at least one of biodegradable materials such as gelatin, collagen, and synthetic peptides because of their adhesiveness to tissues. As the synthetic peptide, a synthetic peptide synthesized organically, a synthetic peptide produced by genetic engineering, or the like can be used. More specifically, in addition to artificial collagen and artificial gelatin imitating natural peptides, peptides having novel physical properties can be used.
　支持体の内部には、凍結乾燥細胞が保持される多数の細孔が形成されている。支持体としては、スポンジ状支持体の他、不織布や織布などを用いることができる。細孔の孔径は、５～１０００μｍであることが好ましく、１０～５００μｍであることがさらに好ましい。細孔の孔径が上記の範囲内であると、例えば、本発明の複合材料を製造しようとして支持体に接触させた、液体培地に細胞を懸濁させた細胞懸濁液が細孔内に進入しやすい。しかも、細孔内に進入した細胞懸濁液のうち、細胞は細孔から流出しにくいが、液体培地は容易に流出するために好ましい。 A large number of pores for holding freeze-dried cells are formed inside the support. As the support, in addition to a sponge-like support, non-woven fabric, woven fabric, or the like can be used. The pore diameter is preferably 5 to 1000 μm, more preferably 10 to 500 μm. When the pore diameter is within the above range, for example, a cell suspension obtained by suspending cells in a liquid medium that has been brought into contact with a support to produce the composite material of the present invention enters the pores. It's easy to do. Moreover, of the cell suspension that has entered the pores, the cells are unlikely to flow out of the pores, but the liquid medium is preferable because it flows out easily.
　本発明の複合材料は、培養細胞や損傷組織の細胞を有効に活性化させてその増殖や分化誘導を促進させるために用いられる。このため、支持体の形状維持期間は、細胞の増殖や分化誘導が完了するまでの期間（ｉｎ　ｖｉｖｏであれば、患部の治療や修復が完了するまでの期間）であることが好ましい。 The composite material of the present invention is used for effectively activating cultured cells and damaged tissue cells to promote their proliferation and differentiation induction. For this reason, the shape maintaining period of the support is preferably a period until cell proliferation and differentiation induction are completed (in vivo, a period until treatment and repair of the affected area are completed).
　支持体を構成する生体親和性材料として生体内分解性材料を用いる場合、支持体の形状維持期間は、生体内分解性材料に分子間架橋を導入すること等によって調整することができる。生体内分解性材料がタンパク質である場合には、化学的架橋、物理的架橋、又は酵素を用いた架橋などによって架橋することができる。化学的架橋に際しては、ホルマリン、グルタルアルデヒド、エチレングリコールジグリシジルエーテル、ポリエチレングリコールジグリシジルエーテル、ポリグリセロールポリグリシジルエーテル、グリセロールポリグリシジルエーテル、ヘキサメチレンジイソシアネート等の化学架橋剤を用いることができる。物理的架橋に際しては、γ線、紫外線等を用いることができる。また、酵素を用いた架橋に際しては、トランスグルタミナーゼ等の酵素を用いることができる。一方、生体内分解性材料が多糖類である場合には、上記の化学架橋剤の他、カルボジイミド等の脱水剤や、Ｃａ塩等を用いて架橋することができる。 When a biodegradable material is used as the biocompatible material constituting the support, the shape maintaining period of the support can be adjusted by introducing intermolecular crosslinks into the biodegradable material. When the biodegradable material is a protein, it can be crosslinked by chemical crosslinking, physical crosslinking, or crosslinking using an enzyme. In the chemical crosslinking, a chemical crosslinking agent such as formalin, glutaraldehyde, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether, glycerol polyglycidyl ether, hexamethylene diisocyanate can be used. For physical crosslinking, γ rays, ultraviolet rays, or the like can be used. In addition, an enzyme such as transglutaminase can be used for crosslinking using an enzyme. On the other hand, when the biodegradable material is a polysaccharide, it can be crosslinked using a dehydrating agent such as carbodiimide, a Ca salt, or the like in addition to the chemical crosslinking agent.
　支持体の大きさ及び形状については特に限定されず、用途に応じた大きさ及び形状とすればよい。支持体の形状の具体例としては、立方体、円柱、シート状などを挙げることができる。なお、シート状の不織布又は織布を巻いて所望の形状にしてもよい。 大 き The size and shape of the support are not particularly limited, and may be a size and a shape corresponding to the application. Specific examples of the shape of the support include a cube, a cylinder, and a sheet. In addition, you may make a desired shape by winding a sheet-like nonwoven fabric or woven fabric.
（凍結乾燥細胞）
　凍結乾燥細胞は、多孔性の支持体の内部に担持させた細胞を凍結乾燥させることによって形成される。細胞としては、例えば、非ヒト胚幹細胞株、ヒト胚幹細胞株、誘導多能性幹細胞、精原幹細胞、胚幹細胞、間葉系幹細胞、内皮幹細胞、造血幹細胞、神経幹細胞、神経冠幹細胞、胃腸幹細胞等の幹細胞；骨髄、脂肪組織、胎盤組織、臍帯組織、臍帯血、及び歯髄などの組織に由来する間葉系幹細胞；ヒト皮膚線維芽細胞、内皮細胞、胃腸の上皮細胞、乳腺上皮細胞、メラノサイト、肺上皮細胞、腎上皮細胞、角化細胞、尿路上皮細胞、肝細胞、角膜細胞、レンズ細胞、骨芽細胞、骨細胞、象牙芽細胞、軟骨細胞、靭帯細胞、腱細胞、脳下垂体細胞、唾液腺細胞、副腎細胞、外分泌腺膵細胞、内分泌腺膵細胞、ライディッヒ細胞、脂肪細胞、線維芽細胞、網膜細胞、ドーパミン作動性ニューロン、ＧＡＢＡ作動性ニューロン、グルタミン作動性ニューロン、コリン作動性ニューロン、アドレナリン作動性ニューロン、星状細胞、乏突起膠細胞、シュワン細胞、平滑筋細胞、平滑筋芽細胞、骨格筋細胞、骨格筋芽細胞、心筋細胞、Ｂリンパ球、Ｔリンパ球、マクロファージ、好中性、樹状細胞、肥満細胞、好酸球、好塩基球、ナチュラルキラー細胞、Ｍ細胞、マイクログリア等の体細胞；遺伝子組換え細胞；ＨｅＬａ、ＨｅＬａＳ３、ＣＣＦ－ＳＴＴＧ１、ＨｅｐＧ２、ＳＫ－ＭＥＬ－５、Ｓａｏｓ－２、ＷＥＲＩ－Ｒｂ－１等の癌細胞株；ＮｕＬｉ、ＣｕＦｉ、ＣＨＯＮ－００１、ＢＪ－５ｔａ、ｈＴＥＲＴ－ＨＭＥ１（ＭＥ１６Ｃ）、ｈＴＥＲＴ－ＨＰＮＥ、ｈＴＥＲＴ　ＲＰＥ－１、ＮｅＨｅｐＬｘＨＴ、ＨＥＳＣ、ＨＯＳ細胞、ＯＵＭＳ２７、ＮＢ－１、Ｔ９８Ｇ、ＫＰ－Ｎ－ＲＴ－ＢＭ－１等の不死化細胞株等を挙げることができる。 (Freeze-dried cells)
Freeze-dried cells are formed by freeze-drying the cells carried inside the porous support. Examples of cells include non-human embryonic stem cell lines, human embryonic stem cell lines, induced pluripotent stem cells, spermatogonial stem cells, embryonic stem cells, mesenchymal stem cells, endothelial stem cells, hematopoietic stem cells, neural stem cells, neural crest stem cells, and gastrointestinal stem cells. Stem cells such as bone marrow, adipose tissue, placental tissue, umbilical cord tissue, umbilical cord blood, and dental pulp; human dermal fibroblasts, endothelial cells, gastrointestinal epithelial cells, mammary epithelial cells, melanocytes , Lung epithelial cells, renal epithelial cells, keratinocytes, urothelial cells, hepatocytes, corneal cells, lens cells, osteoblasts, bone cells, odontoblasts, chondrocytes, ligament cells, tendon cells, pituitary gland Cells, salivary gland cells, adrenal cells, exocrine pancreatic cells, endocrine pancreatic cells, Leydig cells, adipocytes, fibroblasts, retinal cells, dopaminergic neurons, GABAergic neurons, glutamatergic Uron, cholinergic neuron, adrenergic neuron, astrocyte, oligodendrocyte, Schwann cell, smooth muscle cell, smooth myoblast, skeletal muscle cell, skeletal myoblast, cardiomyocyte, B lymphocyte, T Somatic cells such as lymphocytes, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, natural killer cells, M cells, microglia; genetically modified cells; HeLa, HeLaS3, CCF-STTG1 , HepG2, SK-MEL-5, Saos-2, WERI-Rb-1, and other cancer cell lines; NuLi, CuFi, CHON-001, BJ-5ta, hTERT-HME1 (ME16C), hTERT-HPNE, hTERT RPE- 1, NeHepLxHT, HESC, HOS cell, OUMS27, NB-1, T98G, KP-NR Immortalized such as -BM-1 cell lines, and the like can be given.
（その他の成分）
　本発明の複合材料は、凍結乾燥細胞及び細胞内成分以外の成分（その他の成分）が支持体の細孔内にさらに担持されていてもよい。その他の成分としては、培地含有成分、ヒアルロン酸、ヒアルロン酸の化学修飾物、硫酸鉄（ＦｅＳＯ4）、コンドロイチン硫酸、及び各種のサイトカインなどを挙げることができる。これらの成分が支持体の細孔内にさらに担持された複合材料を用いることで、免疫寛容性と恒常性を維持することができる。 (Other ingredients)
In the composite material of the present invention, components other than freeze-dried cells and intracellular components (other components) may be further supported in the pores of the support. Examples of other components include a medium-containing component, hyaluronic acid, a chemically modified product of hyaluronic acid, iron sulfate (FeSO 4 ), chondroitin sulfate, and various cytokines. By using a composite material in which these components are further supported in the pores of the support, immunological tolerance and homeostasis can be maintained.
（細胞活性化用複合材料）
　本発明の複合材料の構成する多孔性の支持体については、プリオンなどの汚染リスクのない飼育管理された動物から採取された原料や、汚染フリーの合成材料を用いて製造されたものであることが好ましい。さらに、本発明の複合材料は、生菌を実質的に含まないものであることが好ましい。生菌を実質的に含まないことで、培養細胞の培養や、細胞や組織が欠損等している生体（ヒト、又はヒト以外の動物など）内の部位への移植により好適に用いることができる。なお、本明細書における「生菌を実質的に含まない」とは、日本薬局方一般試験法に規定する「無菌試験法」に準じて試験した場合に、微生物（細菌又は真菌等）が検出されないことを意味する。生菌を実質的に含まない複合材料を製造するには、例えば、無菌的な操作で複合材料を製造すればよい。 (Composite material for cell activation)
The porous support constituting the composite material of the present invention is manufactured using a raw material collected from a breeding-managed animal such as prion or a contamination-free synthetic material. Is preferred. Furthermore, the composite material of the present invention is preferably substantially free of viable bacteria. By substantially not containing viable bacteria, it can be suitably used for culturing cultured cells and transplanting to a site in a living body (such as a human or non-human animal) in which cells or tissues are defective. . In this specification, “substantially free of viable bacteria” means that microorganisms (bacteria or fungi, etc.) are detected when tested according to the “sterility test method” defined in the Japanese Pharmacopoeia General Test Method. Means not. In order to produce a composite material substantially free of viable bacteria, for example, the composite material may be produced by an aseptic operation.
（細胞活性化用複合材料の使用方法）
　次に、本発明の複合材料の使用方法について説明する。ｉｎ　ｖｉｔｒｏでは、例えば、（ｉ）細胞を培養している培地中に複合材料を配置する、或いは（ｉｉ）透過性を有する膜を備えたチャンバーに複合材料をのせ、細胞を培養している培地中に複合材料を浮かせた状態で配置する。本発明の複合材料をこのように用いることで、複合材料から放出される各種の因子を培養細胞に有効に作用させ、培養細胞の増殖や分化誘導を促進させることができる。 (How to use composite material for cell activation)
Next, a method for using the composite material of the present invention will be described. In vitro, for example, (i) a medium in which a composite material is placed in a medium in which cells are cultured, or (ii) a medium in which cells are cultured by placing the composite material in a chamber having a permeable membrane. Arrange the composite material in a floating state. By using the composite material of the present invention in this way, various factors released from the composite material can be effectively acted on the cultured cells, and the proliferation and differentiation induction of the cultured cells can be promoted.
　また、ｉｎ　ｖｉｖｏでは、例えば、細胞や組織が欠損等している生体（ヒト、又はヒト以外の動物など）内の部位に複合材料を移植する。本発明の複合材料をこのように用いることで、複合材料から放出される各種の因子を周囲の細胞に有効に作用させ、細胞の増殖や分化誘導を促進させて治癒を促進させることができる。本発明の複合材料は、培養細胞そのものではなく、細胞の増殖や分化誘導を促進させる各種の因子を利用するものである。このため、本発明の複合材料は、いわゆる再生医療の規制対象外のものであり、広く普及させることが容易である。 In vivo, for example, a composite material is transplanted into a site in a living body (such as a human or a non-human animal) in which cells or tissues are defective. By using the composite material of the present invention in this way, various factors released from the composite material can be effectively acted on surrounding cells, and cell proliferation and differentiation induction can be promoted to promote healing. The composite material of the present invention utilizes various factors that promote cell proliferation and differentiation induction, not the cultured cells themselves. For this reason, the composite material of the present invention is not subject to so-called regenerative medicine regulation and can be easily spread widely.
＜細胞活性化用複合材料の製造方法＞
　次に、本発明の細胞活性化用複合材料の製造方法について説明する。本発明の細胞活性化用複合材料の製造方法は、生体親和性材料からなる多孔性の支持体の細孔内に細胞を担持させて複合材料前駆体を得る工程（工程（１））と、得られた複合材料前駆体を凍結乾燥する工程（工程（２））とを有する。以下、その詳細について説明する。 <Method for producing composite material for cell activation>
Next, the manufacturing method of the composite material for cell activation of this invention is demonstrated. The method for producing a composite material for cell activation of the present invention includes a step of obtaining a composite material precursor by supporting cells in pores of a porous support made of a biocompatible material (step (1)), A step of freeze-drying the obtained composite material precursor (step (2)). The details will be described below.
　工程（１）では、前述の多孔性の支持体の細孔内に細胞を担持させて複合材料前駆体を得る。支持体の細孔内に細胞を担持させるには、例えば、液体培地に細胞を懸濁させた細胞懸濁液を支持体に接触させ、細孔内に細胞懸濁液を進入させ（染み込ませ）ればよい。これにより、支持体の細孔内に細胞が担持された複合材料前駆体を得ることができる。なお、複合材料前駆体中の細胞の数は、支持体の見かけ体積１２５ｍｍ3当たり、１０4個以上とすることが好ましく、１０7個以上とすることがさらに好ましい。 In the step (1), cells are supported in the pores of the porous support described above to obtain a composite material precursor. In order to support the cells in the pores of the support, for example, a cell suspension in which cells are suspended in a liquid medium is brought into contact with the support, and the cell suspension enters (soaks) into the pores. ) Thereby, the composite material precursor by which the cell was carry | supported in the pore of a support body can be obtained. The number of cells in the composite material precursor is preferably 10 4 or more, and more preferably 10 7 or more, per apparent volume of 125 mm 3 of the support.
　工程（２）では、上記工程（１）で得られた複合材料前駆体を凍結乾燥する。これにより、本発明の細胞活性化用複合材料を得ることができる。なお、支持体の細孔内に細胞を進入させた後、所定の時間が経過してから凍結乾燥することが好ましい。すなわち、支持体の細孔内に細胞を進入させてから所定の時間が経過するまで放置することで、支持体の細孔の内壁に細胞を有効に接着させることができる。所定の時間としては、短時間が好ましく、６０分以内とすることが好ましい。６０分を超えると、細胞に望ましくない影響が現れる場合がある。 In step (2), the composite material precursor obtained in step (1) is freeze-dried. Thereby, the composite material for cell activation of this invention can be obtained. In addition, it is preferable to freeze-dry after predetermined time passes, after making a cell approach into the pore of a support body. That is, the cells can be effectively adhered to the inner walls of the pores of the support by allowing the cells to enter the pores of the support and leaving them for a predetermined time. The predetermined time is preferably a short time, and is preferably within 60 minutes. If it exceeds 60 minutes, undesirable effects may appear on the cells.
　細胞を凍結乾燥する際に一般的に用いられるジメチルスルホキシド（ＤＭＳＯ）やグリセリンなどの水溶性の凍結乾燥保護剤を用いることなく、複合材料前駆体を凍結乾燥することができる。なお、凍結乾燥保護剤を用いずに凍結乾燥することで、得られる複合材料から凍結乾燥保護剤を除去する等の手間を省くことができる。また、複合材料前駆体を－５０℃以下で凍結することが好ましく、－８０℃以下で凍結することがさらに好ましい。より低い温度条件下で複合材料前駆体を凍結してから乾燥することで、増殖や分化誘導を促進させる能力により優れた複合材料を得ることができる。 The composite material precursor can be freeze-dried without using a water-soluble freeze-drying protective agent such as dimethyl sulfoxide (DMSO) or glycerin that is generally used for freeze-drying cells. In addition, it is possible to save the trouble of removing the freeze-drying protective agent from the composite material obtained by freeze-drying without using the freeze-drying protective agent. The composite material precursor is preferably frozen at −50 ° C. or lower, and more preferably at −80 ° C. or lower. By freezing the composite material precursor under a lower temperature condition and then drying it, a composite material superior in ability to promote proliferation and differentiation induction can be obtained.
　凍結乾燥は、例えば、一般的なフリーザーの他、ディープフリーザーなどの極低温フリーザーを使用して実施することができる。また、炭酸ガス（－７８．５℃）、液体窒素（－１９６℃）、液体ヘリウム（－２６９℃）などの冷却媒体（液化ガス）を用いて複合材料前駆体を凍結させてから乾燥することが好ましい。なかでも、窒素やヘリウムなどの不活性ガスの液化ガスを用いて凍結乾燥することが、細胞に対する影響が少ないとともに、安全性も高いために好ましい。なお、段階的に温度を下げて凍結してもよく、一度に温度を下げて凍結してもよいが、一度に温度を下げて複合材料前駆体を凍結することが、増殖や分化誘導を促進させる能力により優れた複合材料を得ることができるために好ましい。具体的には、細胞膜が破れることなく元の形状が維持された凍結細胞の割合を上げることができるので、増殖や分化誘導を促進させる能力がより高い複合材料となる。凍結後は、直ちに減圧下で水を昇華させて乾燥することが好ましく、昇華による乾燥中に複合材料前駆体が凍結していればよい。凍結乾燥機を用いて乾燥してもよく、凍結後に素早く密閉容器内に入れ、減圧して乾燥してもよい。 Freeze-drying can be performed using, for example, a general freezer or a cryogenic freezer such as a deep freezer. Also, the composite material precursor is frozen using a cooling medium (liquefied gas) such as carbon dioxide (−78.5 ° C.), liquid nitrogen (−196 ° C.), liquid helium (−269 ° C.), and then dried. Is preferred. In particular, lyophilization using an inert gas liquefied gas such as nitrogen or helium is preferable because it has little influence on cells and high safety. In addition, it may be frozen by lowering the temperature step by step, or it may be frozen by lowering the temperature at once, but freezing the precursor of the composite material by lowering the temperature at once promotes proliferation and differentiation induction. It is preferable because a composite material superior in ability to be obtained can be obtained. Specifically, since the ratio of frozen cells whose original shape is maintained without breaking the cell membrane can be increased, the composite material has a higher ability to promote proliferation and differentiation induction. After freezing, it is preferable to immediately sublimate water under reduced pressure and dry, as long as the composite material precursor is frozen during drying by sublimation. It may be dried using a freeze dryer, or may be quickly put into a sealed container after freezing and dried under reduced pressure.
＜細胞培養キット＞
　本発明の細胞培養キットは、上述の本発明の細胞活性化用複合材料を備える。上述の複合材料は、培養細胞に有効に作用し、培養細胞を活性化させて増殖を促進させることができる。このため、この複合材料を用いることで、これまで増殖速度が遅く、培養が困難であった各種培養細胞を培養することが可能な細胞培養キットを構成することができる。 <Cell culture kit>
The cell culture kit of the present invention comprises the above-described composite material for cell activation of the present invention. The above-described composite material can effectively act on cultured cells and activate the cultured cells to promote proliferation. For this reason, by using this composite material, it is possible to construct a cell culture kit capable of culturing various cultured cells that have been slow to grow and difficult to culture.
　本発明の細胞培養キットの構成は、上述の複合材料を備えること以外、一般的な従来の細胞培養キットと同様の構成とすることができる。例えば、図１に示すような、複数のウェル５を有するセルカルチャープレートを培養容器１０として用いることができる。すなわち、この培養容器１０のウェル５内に複合材料を収容することで細胞培養キットとすることができる。なお、ウェルのサイズ（直径）や、１つのセルカルチャープレート当たりのウェルの数などは特に限定されない。ウェルの直径は、例えば、０．６４～３５ｍｍなどである。また、１つのセルカルチャープレート当たりのウェルの数は、例えば、４～９６などである。 The configuration of the cell culture kit of the present invention can be the same as that of a general conventional cell culture kit except that the above-described composite material is provided. For example, a cell culture plate having a plurality of wells 5 as shown in FIG. That is, a cell culture kit can be obtained by accommodating the composite material in the well 5 of the culture vessel 10. In addition, the size (diameter) of a well, the number of wells per one cell culture plate, etc. are not specifically limited. The diameter of the well is, for example, 0.64 to 35 mm. The number of wells per cell culture plate is, for example, 4 to 96.
　また、図２に示すようなシャーレを培養容器２０として用いることもできる。すなわち、この培養容器２０内に複合材料を収容することで細胞培養キットとすることができる。なお、シャーレのサイズ（直径）などは特に限定されない。シャーレの直径は、例えば、３５～１５０ｍｍなどである。セルカルチャープレートやシャーレは、いずれも市販のものを用いることができる。 A petri dish as shown in FIG. 2 can also be used as the culture container 20. That is, a cell culture kit can be obtained by housing the composite material in the culture vessel 20. The size (diameter) of the petri dish is not particularly limited. The diameter of the petri dish is, for example, 35 to 150 mm. Commercially available cell culture plates and petri dishes can be used.
　図１に示す培養容器１０を用いて培養細胞を培養するには、例えば、培養細胞及び培地などをウェル５内に収容するとともに、収容した培養細胞や培地と接触するように複合材料を配置すればよい。また、図２に示す培養容器２０を用いて培養細胞を培養するには、例えば、培養細胞及び培地などを培養容器２０に収容するとともに、収容した培養細胞や培地と接触するように複合材料を配置すればよい。 In order to culture cultured cells using the culture vessel 10 shown in FIG. 1, for example, the cultured cells and the medium are accommodated in the well 5 and a composite material is disposed so as to be in contact with the accommodated cultured cells and medium. That's fine. In order to culture cultured cells using the culture container 20 shown in FIG. 2, for example, the cultured cells and the medium are accommodated in the culture container 20 and the composite material is brought into contact with the accommodated cultured cells and medium. What is necessary is just to arrange.
　図３は、本発明の細胞培養キットの一実施形態を示す模式図である。図３に示す実施形態の細胞培養キット１００は、培養細胞収容部３５と、複合材料収容部４５とを有する培養容器５０を備える。培養細胞収容部３５は、培養細胞３１及び培地３３を収容する部分（空間）である。また、複合材料収容部４５は、複合材料４３を配置する部分（空間）である。複合材料収容部４５に収容された複合材料４３は、複合材料収容部４５の底部を構成する隔膜４７を介在させた状態で、培養細胞収容部３５内に収容された培養細胞３１と間接的に接触する。なお、隔膜４７は、複合材料４３から流出する各種成分や培地成分は透過するが、載置された複合材料４３は下方へと落下しない程度のサイズの孔が形成された膜である。図３に示すような構成とすることで、複合材料から放出される各種因子を、過度の負荷を掛けることなく培養細胞に作用させて、増殖や分化誘導を促進させつつ培養することができる。 FIG. 3 is a schematic view showing one embodiment of the cell culture kit of the present invention. The cell culture kit 100 of the embodiment shown in FIG. 3 includes a culture container 50 having a cultured cell storage unit 35 and a composite material storage unit 45. The cultured cell storage unit 35 is a part (space) that stores the cultured cells 31 and the culture medium 33. The composite material housing 45 is a portion (space) in which the composite material 43 is disposed. The composite material 43 accommodated in the composite material accommodating portion 45 indirectly with the cultured cells 31 accommodated in the cultured cell accommodating portion 35 with the diaphragm 47 constituting the bottom of the composite material accommodating portion 45 interposed therebetween. Contact. The diaphragm 47 is a film in which various components and medium components flowing out from the composite material 43 are permeated, but the placed composite material 43 is formed with a hole of a size that does not fall downward. With the configuration as shown in FIG. 3, various factors released from the composite material can be cultured while acting on cultured cells without applying an excessive load, and promoting proliferation and differentiation induction.
　用いる複合材料のサイズは、セルカルチャープレートのウェルやシャーレの大きさに応じて適宜設定すればよい。複合材料は、ウェル等の大きさを考慮して予め所望とするサイズとなるように製造してもよいし、所望とするサイズとなるように切断等してもよい。 The size of the composite material to be used may be appropriately set according to the size of the well or petri dish of the cell culture plate. The composite material may be manufactured in advance to have a desired size in consideration of the size of a well or the like, or may be cut to have a desired size.
　本発明の細胞培養キットを用いて培養させることが可能な細胞（培養細胞）としては、例えば、前述の凍結乾燥細胞を形成するために用いる細胞と同様の細胞を挙げることができる。なかでも、間葉系幹細胞、プライマリ細胞などの増殖が遅い細胞を培養する場合に特に有効である。 Examples of cells (cultured cells) that can be cultured using the cell culture kit of the present invention include the same cells as those used to form the above-mentioned freeze-dried cells. Among these, it is particularly effective when culturing slow-growing cells such as mesenchymal stem cells and primary cells.
　以下、本発明を実施例に基づいて具体的に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited to these examples.
＜複合材料の製造＞
（実施例１）
　架橋したゼラチンスポンジを直径６ｍｍ、高さ５ｍｍの円柱状に無菌的にカットした。円柱状のゼラチンスポンジの上に、Ｖ７９細胞（繊維芽細胞）の懸濁液（１０6ｃｅｌｌｓ／５０μＬ、ＭＥＭ培地＋２ｍｍｏｌ／Ｌグルタミン）を載置して３０分間放置し、懸濁液をゼラチンスポンジに染み込ませて複合材料前駆体を得た。なお、Ｖ７９細胞としては、ＪＣＲＢ細胞バンクより入手したもの（細胞登録番号ＪＣＲＢ０６０３、Ｌｏｔ　Ｎｏ．１００１２０１２）を用いた。液体窒素を用いて複合材料前駆体を瞬時に凍結させた後、凍結乾燥機（商品名「ＦＤ－５Ｎ」、ＥＹＥＬＡ社製）に移し、直ちに吸引減圧して凍結乾燥し、複合材料を得た。 <Manufacture of composite materials>
Example 1
The cross-linked gelatin sponge was aseptically cut into a cylindrical shape having a diameter of 6 mm and a height of 5 mm. A suspension of V79 cells (fibroblasts) (10 6 cells / 50 μL, MEM medium + 2 mmol / L glutamine) was placed on a cylindrical gelatin sponge and allowed to stand for 30 minutes. So that a composite material precursor was obtained. In addition, as V79 cell, what was obtained from JCRB cell bank (cell registration number JCRB0603, Lot No. 10012012) was used. After instantly freezing the composite material precursor using liquid nitrogen, it was transferred to a lyophilizer (trade name “FD-5N”, manufactured by EYELA Co., Ltd.) and immediately evacuated and lyophilized to obtain a composite material. .
　得られた複合材料の一部を無菌的にカットし、円柱状（直径６ｍｍ、高さ１ｍｍ）の無菌試験用の試料片を２枚作製した。作製した試料片を用いて、日本薬局方一般試験法に規定する「無菌試験法」に準じて無菌試験を行った。具体的には、作製した試料片を、液状チオグリコール酸培地及びソイビーン・カゼイン・ダイジェスト培地にそれぞれ入れ、１４日間培養した。その結果、いずれの培地にも微生物の増殖は観察されなかったため、複合材料は実質的に無菌であると判断した。以下、製造したすべての複合材料について上記と同様の方法で無菌試験を行い、いずれの複合材料も実質的に無菌であることを確認した。 A part of the obtained composite material was cut aseptically to prepare two sample pieces for sterility test in a cylindrical shape (diameter 6 mm, height 1 mm). Using the prepared sample piece, a sterility test was performed according to the “sterility test method” defined in the Japanese Pharmacopoeia General Test Method. Specifically, the prepared sample pieces were placed in a liquid thioglycolic acid medium and a soybean / casein / digest medium, respectively, and cultured for 14 days. As a result, since no growth of microorganisms was observed in any of the media, the composite material was judged to be substantially sterile. Hereinafter, the sterility test was performed on all manufactured composite materials in the same manner as described above, and it was confirmed that all the composite materials were substantially sterile.
（実施例２）
　実施例１で得た複合材料前駆体を、－８０℃のディープフリーザー（品番「ＣＬＮ－３０ＵＷ」、日本フリーザー社製）に入れて凍結させた後、実施例１と同様に凍結乾燥して複合材料を得た。 (Example 2)
The composite material precursor obtained in Example 1 was frozen in a deep freezer (product number “CLN-30UW”, manufactured by Nippon Freezer Co., Ltd.) at −80 ° C. and then freeze-dried in the same manner as in Example 1 to form a composite. Obtained material.
（実施例３）
　実施例１で得た複合材料前駆体を、－２０℃のフリーザー（品番「ＭＤＦ－ＭＵ３００Ｈ－Ｐ」、パナソニックヘルケア社製）に入れた後、さらに液体窒素を用いて段階的に凍結させた。次いで、実施例１と同様に凍結乾燥して複合材料を得た。 (Example 3)
The composite material precursor obtained in Example 1 was placed in a freezer (product number “MDF-MU300H-P”, manufactured by Panasonic Hercare) at −20 ° C., and further frozen in stages using liquid nitrogen. . Subsequently, it lyophilized similarly to Example 1 and the composite material was obtained.
（比較例１）
　実施例１で用いた円柱状のゼラチンスポンジ（凍結乾燥細胞を含まないもの）を比較例１の材料とした。 (Comparative Example 1)
The cylindrical gelatin sponge (without lyophilized cells) used in Example 1 was used as the material of Comparative Example 1.
（評価（１））
　Ｖ７９細胞５０ｃｅｌｌｓを液体培地（ＭＥＭ培地＋２ｍｍｏｌ／Ｌグルタミン＋１０質量％ＦＢＳ（ウシ胎児血清、ライフ　テクノロジーズ社製、Ｌｏｔ　Ｎｏ．Ｓ０９０９９Ｓ１８２０））２ｍＬに懸濁させた。得られた懸濁物を３枚のセルカルチャープレート（２４ウェル）の各ウェルに入れ、炭酸ガスインキュベーター内で１８時間培養した。培養後、複合材料が落下しない程度の大きさの穴を開けたカルチャーインサート（ポアサイズ：８μｍ、Ｆａｌｃｏｎ社製）に、実施例１の複合材料を入れる、実施例２の複合材料を入れる、実施例３の複合材料を入れる、及び何も入れない（コントロール）の４つプレートに分け、炭酸ガスインキュベーター内で６日間培養した。培養後、液体培地を捨て、ホルムアルデヒドで培養細胞のコロニーを固定した。次いで、クリスタルバイオレット（０．５質量％溶液）を用いてコロニーを染色し、コロニー数をカウントして１ウェル当たりの平均値を算出した。そして、コントロールのコロニー数（平均値）を「１００．０％」とする相対値（％）を算出した。結果を図４に示す。 (Evaluation (1))
50 cells of V79 cells were suspended in 2 mL of a liquid medium (MEM medium + 2 mmol / L glutamine + 10 mass% FBS (fetal bovine serum, manufactured by Life Technologies, Lot No. S09099S1820)). The obtained suspension was put into each well of three cell culture plates (24 wells) and cultured for 18 hours in a carbon dioxide incubator. After the culture, the composite material of Example 1 is put into the culture insert (pore size: 8 μm, manufactured by Falcon) with a hole that is large enough not to drop the composite material. Three composite materials were added, and the plate was divided into four plates containing no control (control) and cultured for 6 days in a carbon dioxide incubator. After culturing, the liquid medium was discarded and colonies of cultured cells were fixed with formaldehyde. Subsequently, the colony was dye | stained using crystal violet (0.5 mass% solution), the number of colonies was counted, and the average value per well was computed. And the relative value (%) which made colony number (average value) of control "100.0%" was computed. The results are shown in FIG.
（評価（２））
　実施例１～３の複合材料を導電テープで試料台に張り付けた。イオンスパッタ装置（商品名「Ｅ－１０１」、日立製作所社製）を使用してスパッタリングした後、走査型電子顕微鏡（商品名「Ｓ－４８００」、日立製作所社製）を使用して微細構造を観察した。５００倍に拡大したところ、いずれの複合材料についても、球状の凍結乾燥細胞が５個以上集まった凍結乾燥細胞塊が観察された。また、撮影した電子顕微鏡写真を図５～７に示す。 (Evaluation (2))
The composite materials of Examples 1 to 3 were attached to a sample table with a conductive tape. Sputtering is performed using an ion sputtering apparatus (trade name “E-101”, manufactured by Hitachi, Ltd.), and then a fine structure is formed using a scanning electron microscope (trade name “S-4800”, manufactured by Hitachi, Ltd.). Observed. When the magnification was 500 times, a freeze-dried cell mass in which 5 or more spherical freeze-dried cells gathered was observed for any composite material. The photographed electron micrographs are shown in FIGS.
（評価（３））
　Ｖ７９細胞５０ｃｅｌｌｓを液体培地（ＭＥＭ培地＋２ｍｍｏｌ／Ｌグルタミン＋１０質量％ＦＢＳ（ウシ胎児血清、ライフ　テクノロジーズ社製、Ｌｏｔ　Ｎｏ．Ｓ０９０９９Ｓ１８２０））２ｍＬに懸濁させた。得られた懸濁物を３枚のセルカルチャープレート（２４ウェル）の各ウェルに入れ、炭酸ガスインキュベーター内で１８時間培養した。培養後、各ウェルに、実施例１の複合材料を入れる（評価例１）、比較例１の材料を入れる（評価例２）、及び何も入れない（評価例３）の３つプレートに分け、炭酸ガスインキュベーター内で６日間培養した。培養後、液体培地を捨て、ホルムアルデヒドで培養細胞のコロニーを固定した。次いで、クリスタルバイオレット（０．５質量％溶液）を用いてコロニーを染色し、コロニー数をカウントして１ウェル当たりの平均値を算出した。結果を表１に示す。 (Evaluation (3))
50 cells of V79 cells were suspended in 2 mL of a liquid medium (MEM medium + 2 mmol / L glutamine + 10 mass% FBS (fetal bovine serum, manufactured by Life Technologies, Lot No. S09099S1820)). The obtained suspension was put into each well of three cell culture plates (24 wells) and cultured for 18 hours in a carbon dioxide incubator. After the incubation, the composite material of Example 1 is put into each well (Evaluation Example 1), the material of Comparative Example 1 (Evaluation Example 2), and nothing (Evaluation Example 3). The cells were cultured for 6 days in a carbon dioxide incubator. After culturing, the liquid medium was discarded and colonies of cultured cells were fixed with formaldehyde. Subsequently, the colony was dye | stained using crystal violet (0.5 mass% solution), the number of colonies was counted, and the average value per well was computed. The results are shown in Table 1.
　また、クリスタルバイオレットで染色したコロニーをエタノールで洗浄及び抽出して染色液を得た。得られた染色液をマイクロプレート（９６ウェル）の各ウェルに１．０ｍＬずつ入れ、プレートリーダーを使用して吸光度（５９０ｎｍ）を測定し、１ウェル当たりの平均値を算出した。結果を表１に示す。さらに、増殖率（評価例３の吸光度を「１．００」とする吸光度の相対値）を算出した結果を表１に示す。 Moreover, colonies stained with crystal violet were washed and extracted with ethanol to obtain a staining solution. 1.0 mL of the obtained staining solution was placed in each well of a microplate (96 wells), and the absorbance (590 nm) was measured using a plate reader, and the average value per well was calculated. The results are shown in Table 1. Furthermore, Table 1 shows the results of calculating the growth rate (relative value of the absorbance with the absorbance in Evaluation Example 3 set to “1.00”).
Figure JPOXMLDOC01-appb-I000001
Figure JPOXMLDOC01-appb-I000001
（評価（４））
　Ｖ７９細胞５０ｃｅｌｌｓを液体培地（ＭＥＭ培地＋２ｍｍｏｌ／Ｌグルタミン＋１０質量％ＦＢＳ（ウシ胎児血清、ライフ　テクノロジーズ社製、Ｌｏｔ　Ｎｏ．Ｓ０９０９９Ｓ１８２０））２ｍＬに懸濁させた。得られた懸濁物を３枚のセルカルチャープレート（２４ウェル）の各ウェルに入れ、炭酸ガスインキュベーター内で１８時間培養した。培養後、複合材料が落下しない程度の大きさの穴を開けたカルチャーインサート（ポアサイズ：８μｍ、Ｆａｌｃｏｎ社製）に、実施例１の複合材料を入れる（評価例４）、比較例１の材料を入れる（評価例５）、及び何も入れない（評価例６）の３つプレートに分け、炭酸ガスインキュベーター内で６日間培養した。培養後、液体培地を捨て、ホルムアルデヒドで培養細胞のコロニーを固定した。次いで、クリスタルバイオレット（０．５質量％溶液）を用いてコロニーを染色し、コロニー数をカウントして１ウェル当たりの平均値を算出した。結果を表２に示す。 (Evaluation (4))
50 cells of V79 cells were suspended in 2 mL of a liquid medium (MEM medium + 2 mmol / L glutamine + 10 mass% FBS (fetal bovine serum, manufactured by Life Technologies, Lot No. S09099S1820)). The obtained suspension was put into each well of three cell culture plates (24 wells) and cultured for 18 hours in a carbon dioxide incubator. After culturing, the composite material of Example 1 is put into a culture insert (pore size: 8 μm, manufactured by Falcon) having a hole that does not drop the composite material, and the material of Comparative Example 1 is used. The plate was divided into three plates (Evaluation Example 5) and nothing (Evaluation Example 6) and cultured for 6 days in a carbon dioxide incubator. After culturing, the liquid medium was discarded and colonies of cultured cells were fixed with formaldehyde. Subsequently, the colony was dye | stained using crystal violet (0.5 mass% solution), the number of colonies was counted, and the average value per well was computed. The results are shown in Table 2.
　また、前述の「評価（３）」と同様の操作により得られた染色液をマイクロプレート（９６ウェル）の各ウェルに１．０ｍＬずつ入れ、プレートリーダーを使用して吸光度（５９０ｎｍ）を測定し、１ウェル当たりの平均値を算出した。結果を表２に示す。さらに、増殖率（評価例６の吸光度を「１．００」とする吸光度の相対値）を算出した結果を表２に示す。 In addition, 1.0 mL of the staining solution obtained by the same operation as in the above “evaluation (3)” is put into each well of the microplate (96 wells), and the absorbance (590 nm) is measured using a plate reader. The average value per well was calculated. The results are shown in Table 2. Further, Table 2 shows the results of calculating the growth rate (relative value of absorbance with the absorbance of Evaluation Example 6 being “1.00”).
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000002
（実施例４）
　Ｖ７９細胞（繊維芽細胞）の懸濁液（１０5ｃｅｌｌｓ／５０μＬ、ＭＥＭ培地＋２ｍｍｏｌ／Ｌグルタミン）を用いたこと以外は、前述の実施例１と同様にして複合材料を得た。 Example 4
A composite material was obtained in the same manner as in Example 1 except that a suspension of V79 cells (fibroblasts) (10 5 cells / 50 μL, MEM medium + 2 mmol / L glutamine) was used.
（評価（５））
　上記の実施例４で得た複合材料を使用し、前述の「評価（４）」と同様の評価を行った。結果を表３に示す。 (Evaluation (5))
The composite material obtained in Example 4 was used, and the same evaluation as in the above “Evaluation (4)” was performed. The results are shown in Table 3.
Figure JPOXMLDOC01-appb-I000003
Figure JPOXMLDOC01-appb-I000003
（実施例５）
　架橋したゼラチンスポンジを直径６ｍｍ、高さ５ｍｍの円柱状に無菌的にカットした。円柱状のゼラチンスポンジの上に、ｈＭＳＣ細胞（ヒト間葉系幹細胞）の懸濁液（１０6ｃｅｌｌｓ／５０μＬ、ＭＥＭ培地＋２ｍｍｏｌ／Ｌグルタミン）を載置して３０分間放置し、懸濁液をゼラチンスポンジに染み込ませて複合材料前駆体を得た。なお、ｈＭＳＣ細胞としては、Ｌｏｎｚａ社より入手したものを用いた。液体窒素を用いて複合材料前駆体を瞬時に凍結させた後、凍結乾燥機に移し、直ちに吸引減圧して凍結乾燥し、複合材料を得た。 (Example 5)
The cross-linked gelatin sponge was aseptically cut into a cylindrical shape having a diameter of 6 mm and a height of 5 mm. A suspension of hMSC cells (human mesenchymal stem cells) (10 6 cells / 50 μL, MEM medium + 2 mmol / L glutamine) was placed on a cylindrical gelatin sponge and allowed to stand for 30 minutes. A composite material precursor was obtained by soaking into a gelatin sponge. As hMSC cells, those obtained from Lonza were used. The composite material precursor was instantly frozen using liquid nitrogen, then transferred to a lyophilizer, and immediately evacuated and vacuum-dried to obtain a composite material.
（比較例２）
　実施例５で用いた円柱状のゼラチンスポンジ（凍結乾燥細胞を含まないもの）を比較例２の材料とした。 (Comparative Example 2)
The cylindrical gelatin sponge (without lyophilized cells) used in Example 5 was used as the material of Comparative Example 2.
（実施例６）
　ｈＭＳＣ細胞（ヒト間葉系幹細胞）の懸濁液（１０5ｃｅｌｌｓ／５０μＬ、ＭＥＭ培地＋２ｍｍｏｌ／Ｌグルタミン）を用いたこと以外は、前述の実施例５と同様にして複合材料を得た。 (Example 6)
A composite material was obtained in the same manner as in Example 5 except that a suspension of hMSC cells (human mesenchymal stem cells) (10 5 cells / 50 μL, MEM medium + 2 mmol / L glutamine) was used.
（評価（６））
　ｈＭＳＣ細胞４×１０3ｃｅｌｌｓを液体培地（Ｌｏｎｚａ社製、ＭＳＣＧＭ　Ｂｕｌｌｅｔｋｉｔ）２ｍＬに懸濁させた。得られた懸濁物を４枚のセルカルチャープレート（２４ウェル）の各ウェルに入れ、炭酸ガスインキュベーター内で１８時間培養した。培養後、複合材料が落下しない程度の大きさの穴を開けたカルチャーインサート（ポアサイズ：８μｍ、Ｆａｌｃｏｎ社製）に、実施例５の複合材料を入れる（評価例１１）、比較例２の材料を入れる（評価例１２）、実施例６の複合材料を入れる（評価例１３）、及び何も入れない（評価例１４）の４つプレートに分け、炭酸ガスインキュベーター内で６日間培養した。生細胞数測定試薬（商品名「ＷＳＴ－８」（Ｃｅｌｌ　Ｃｏｕｎｔ　ｒｅａｇｅｎｔ　ＳＦ）、半井化学社製）を使用して細胞数をカウントして１ウェル当たりの平均値を算出した。結果を表４に示す。 (Evaluation (6))
hMSC cells 4 × 10 3 cells were suspended in 2 mL of liquid medium (Lonza, MSCGM Bulletkit). The obtained suspension was placed in each well of four cell culture plates (24 wells) and cultured for 18 hours in a carbon dioxide incubator. After culturing, the composite material of Example 5 is put into a culture insert (pore size: 8 μm, manufactured by Falcon) having a hole that does not drop the composite material (Evaluation Example 11), and the material of Comparative Example 2 is used. The plate was divided into four plates (Evaluation Example 12), the composite material of Example 6 (Evaluation Example 13), and nothing (Evaluation Example 14), and cultured in a carbon dioxide incubator for 6 days. The number of cells was counted using a reagent for measuring the number of living cells (trade name “WST-8” (Cell Count reagent SF), manufactured by Hanai Chemical Co., Ltd.), and the average value per well was calculated. The results are shown in Table 4.
　また、前述の「評価（３）」と同様の操作により得られた染色液をマイクロプレート（９６ウェル）の各ウェルに１．０ｍＬずつ入れ、プレートリーダーを使用して吸光度（４５０ｎｍ）を測定し、１ウェル当たりの平均値を算出した。結果を表４に示す。さらに、増殖率（評価例１４の吸光度を「１．００」とする吸光度の相対値）を算出した結果を表４に示す。 In addition, 1.0 mL of the staining solution obtained by the same operation as in the above “evaluation (3)” is put into each well of the microplate (96 wells), and the absorbance (450 nm) is measured using a plate reader. The average value per well was calculated. The results are shown in Table 4. Furthermore, Table 4 shows the results of calculating the growth rate (relative value of absorbance with the absorbance of Evaluation Example 14 as “1.00”).
Figure JPOXMLDOC01-appb-I000004
Figure JPOXMLDOC01-appb-I000004
　本発明の細胞活性化用複合材料は、培養細胞や損傷組織の細胞を有効に活性化させ、その増殖や分化誘導を促進させるための材料として有用である。 The composite material for cell activation of the present invention is useful as a material for effectively activating cultured cells and cells of damaged tissues and promoting their proliferation and differentiation induction.
５：ウェル
１０，２０，５０：培養容器
３１：培養細胞
３３：培地
３５：培養細胞収容部
４３：複合材料
４５：複合材料収容部
４７：隔膜
１００：細胞培養キット 5: Well 10, 20, 50: Culture vessel 31: Cultured cell 33: Medium 35: Cultured cell container 43: Composite material 45: Composite material container 47: Diaphragm 100: Cell culture kit

Claims (13)

  1. 　生体親和性材料からなる多孔性の支持体の内部に細胞を担持させた複合材料前駆体を凍結乾燥して得られる細胞活性化用複合材料。 A composite material for cell activation obtained by freeze-drying a composite material precursor in which cells are supported inside a porous support made of a biocompatible material.
  2. 　前記支持体と、前記支持体の細孔内に担持された、前記細胞が凍結乾燥した凍結乾燥細胞と、を含む請求項１に記載の細胞活性化用複合材料。 2. The composite material for cell activation according to claim 1, comprising the support and freeze-dried cells on which the cells are lyophilized and supported in the pores of the support.
  3. 　前記支持体の前記細孔内に担持された、培地含有成分、ヒアルロン酸、ヒアルロン酸の化学修飾物、硫酸鉄（ＦｅＳＯ4）、コンドロイチン硫酸、及びサイトカインからなる群より選択される少なくとも一種をさらに含む請求項２に記載の細胞活性化用複合材料。 At least one selected from the group consisting of a medium-containing component, hyaluronic acid, a chemically modified product of hyaluronic acid, iron sulfate (FeSO 4 ), chondroitin sulfate, and a cytokine supported in the pores of the support; The composite material for cell activation according to claim 2 comprising.
  4. 　前記生体親和性材料が、ゼラチン、コラーゲン、及び合成ペプチドの少なくともいずれかの生体内分解性材料である請求項１～３のいずれか一項に記載の細胞活性化用複合材料。 The composite material for cell activation according to any one of claims 1 to 3, wherein the biocompatible material is a biodegradable material of at least one of gelatin, collagen, and a synthetic peptide.
  5. 　前記支持体がスポンジ状支持体である請求項１～４のいずれか一項に記載の細胞活性化用複合材料。 The composite material for cell activation according to any one of claims 1 to 4, wherein the support is a sponge-like support.
  6. 　前記細胞が、間葉系幹細胞、体細胞、遺伝子組み換え細胞、癌細胞株、及び不死化細胞株からなる群より選択される少なくとも一種である請求項１～５のいずれか一項に記載の細胞活性化複合材料。 The cell according to any one of claims 1 to 5, wherein the cell is at least one selected from the group consisting of mesenchymal stem cells, somatic cells, genetically modified cells, cancer cell lines, and immortalized cell lines. Activated composite material.
  7. 　生菌を実質的に含まない請求項１～６のいずれか一項に記載の細胞活性化用複合材料。 The composite material for cell activation according to any one of claims 1 to 6, which is substantially free of viable bacteria.
  8. 　内部の任意の領域を走査型電子顕微鏡により５００倍に拡大して観察した場合に、球状の前記凍結乾燥細胞が５個以上集まった凍結乾燥細胞塊が観察される請求項２～７のいずれか一項に記載の細胞活性化用複合材料。 8. A freeze-dried cell mass in which five or more spherical freeze-dried cells are collected when an arbitrary region inside is observed with a scanning electron microscope at a magnification of 500 times. The composite material for cell activation according to one item.
  9. 　請求項１～８のいずれか一項に記載の細胞活性化用複合材料の製造方法であって、
    　生体親和性材料からなる多孔性の支持体の細孔内に細胞を担持させて複合材料前駆体を得る工程（１）と、
    　得られた前記複合材料前駆体を凍結乾燥する工程（２）と、を有する細胞活性化用複合材料の製造方法。     A method for producing a composite material for cell activation according to any one of claims 1 to 8,
    A step (1) of obtaining a composite material precursor by supporting cells in the pores of a porous support made of a biocompatible material;
    A step (2) of freeze-drying the obtained composite material precursor, and a method for producing a composite material for cell activation.
  10. 　前記工程（２）における、前記複合材料前駆体を凍結する温度が－５０℃以下である請求項９に記載の細胞活性化用複合材料の製造方法。 The method for producing a composite material for cell activation according to claim 9, wherein the temperature at which the composite material precursor is frozen in the step (2) is -50 ° C or lower.
  11. 　請求項１～８のいずれか一項に記載の細胞活性化用複合材料を備えた細胞培養キット。 A cell culture kit comprising the composite material for cell activation according to any one of claims 1 to 8.
  12. 　培養容器をさらに備え、
    　前記培養容器は、培養細胞を収容する培養細胞収容部と、前記培養細胞収容部内に収容された前記培養細胞と直接的又は間接的に接触する位置に前記細胞活性化用複合材料を配置する複合材料収容部と、を有する請求項１１に記載の細胞培養キット。     A culture vessel,
    The culture container is a composite in which the cell activation composite material is disposed at a position where the culture cell storage unit stores the culture cell, and the culture cell stored in the culture cell storage unit directly or indirectly contacts the culture cell storage unit. The cell culture kit according to claim 11, comprising a material container.
  13. 　培養細胞を培養するための、請求項１～８のいずれか一項に記載の細胞活性化用複合材料の使用。 Use of the composite material for cell activation according to any one of claims 1 to 8 for culturing cultured cells.
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JP2020110114A (en) * 2019-01-15 2020-07-27 東レ・メディカル株式会社 Cell culture insert

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WO1996015818A1 (en) * 1994-11-22 1996-05-30 Tissue Engineering, Inc. Biopolymer foams having extracellular matrix particulates
JP2008136396A (en) * 2006-11-30 2008-06-19 Yokohama City Univ Method for culturing cartilage cell

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WO1996015818A1 (en) * 1994-11-22 1996-05-30 Tissue Engineering, Inc. Biopolymer foams having extracellular matrix particulates
JP2008136396A (en) * 2006-11-30 2008-06-19 Yokohama City Univ Method for culturing cartilage cell

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Publication number Priority date Publication date Assignee Title
JP2020110114A (en) * 2019-01-15 2020-07-27 東レ・メディカル株式会社 Cell culture insert
JP7265243B2 (en) 2019-01-15 2023-04-26 ネッパジーン株式会社 cell culture insert

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