WO2006123658A1 - Cell culture substrate, culture apparatus and cell culture method - Google Patents

Cell culture substrate, culture apparatus and cell culture method Download PDF

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Publication number
WO2006123658A1
WO2006123658A1 PCT/JP2006/309746 JP2006309746W WO2006123658A1 WO 2006123658 A1 WO2006123658 A1 WO 2006123658A1 JP 2006309746 W JP2006309746 W JP 2006309746W WO 2006123658 A1 WO2006123658 A1 WO 2006123658A1
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Prior art keywords
cell culture
cells
culture
substrate
cell
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PCT/JP2006/309746
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French (fr)
Japanese (ja)
Inventor
Yasuyuki Ishizuka
Tetsuro Ogawa
Tsuyoshi Ishikawa
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Applied Cell Biotechnologies, Inc.
Pentax Corporation
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Application filed by Applied Cell Biotechnologies, Inc., Pentax Corporation filed Critical Applied Cell Biotechnologies, Inc.
Publication of WO2006123658A1 publication Critical patent/WO2006123658A1/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
    • 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
    • C12N2533/10—Mineral substrates
    • C12N2533/18—Calcium salts, e.g. apatite, Mineral components from bones, teeth, shells

Definitions

  • the present invention relates to a substrate used for culturing with cells attached thereto, a culture instrument equipped with a cell culture substrate, and a cell culture method.
  • Cells are roughly classified into suspension cells and adherent cells. Suspended cells are cells that do not require a scaffold for growth and proliferation, and adherent cells are cells that require a scaffold for growth and proliferation. Most stem cells important in regenerative medicine are adherent cells, and their culture requires attachment of cells based on artificially prepared scaffolds.
  • a scaffold for adherent cells synthetic polymers such as polystyrene, biological proteins such as collagen, and the like are used.
  • various improvements have been made when cell adhesion to a base material serving as a scaffold is increased or proteins are easily adsorbed to the base material serving as a scaffold.
  • bioabsorbable materials such as PGA (polyglycolic acid) as a scaffold base material.
  • PGA polyglycolic acid
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2005-27532 describes a cell culture substrate comprising a hydrophilic polymer gel containing fine particles and a hydrophilic component.
  • this cell culture substrate for example, fine particles, a monomer of a hydrophilic component, and a crosslinking agent are uniformly dispersed in a solvent, and then the monomer is polymerized.
  • the fine particles include organic fine particles such as polystyrene, and inorganic fine particles such as silica, silica and idoxyapatite.
  • Patent Document 2 Japanese Patent Laid-Open No. 2001-157574 describes a cell culture substrate comprising a honeycomb structure containing a biodegradable polymer.
  • a solution containing a biodegradable polymer and an amphiphilic polymer and a hydrophobic organic solvent is cast on a substrate in an atmosphere having a relative humidity of 50 to 95%, and the hydrophobic organic solvent is obtained.
  • the water evaporates gradually, condensation occurs on the surface of the casting liquid, and the water droplets generated by the condensation evaporate.
  • a hydrophobic organic solvent is indispensable for the production of this honeycomb structure, and there seems to be a problem from the viewpoint of safety.
  • Patent Document 3 Japanese Patent Laid-Open No. 2005-522244 describes a bone regeneration base material in which a bioabsorbable organic porous material having communication holes and a bone-compatible inorganic material are combined. .
  • a bioabsorbable organic porous material having communication holes and a bone-compatible inorganic material are combined.
  • this bone regeneration A substrate for use can be obtained.
  • the form of the porous body include fibers, woven sheets, and non-woven fabrics.
  • the bone-friendly inorganic material is simply filled in the communicating holes of the bioabsorbable organic porous material and contributes to bone formation. sell.
  • the bone-friendly inorganic material is filled in the communicating holes of the bioabsorbable organic porous material in order to contribute to cell growth. I understood that.
  • the base material in which the communicating holes are filled with the bone-compatible inorganic material does not have an appropriate space for the cells to grow, so that it is suitable for culture.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2005-27532
  • Patent Document 2 Japanese Patent Laid-Open No. 2001-157574
  • Patent Document 3 Japanese Patent Laid-Open No. 2005-52224
  • An object of the present invention is to provide a cell culturing means capable of generating cells that do not adhere to a substrate and a cell culturing method using such a cell culturing means.
  • the cell culture substrate of the present invention is sheet-shaped and is characterized by comprising calcium phosphate and fibers.
  • the cell culture substrate is preferably covered with calcium phosphate.
  • the fiber preferably forms a nonwoven fabric, and the weight per unit area of the nonwoven fabric is preferably 0.1 to 0.3 g / 100 cm 2 .
  • the calcium phosphate is preferably hydroxyapatite.
  • a cell culture instrument of the present invention comprises the cell culture substrate of the present invention.
  • the cell culture method of the present invention is characterized by using the cell culture substrate of the present invention or the culture instrument of the present invention.
  • the cells adhere to each other, so that the cells can be proliferated even in a portion where the cells are not in contact with the cell culture substrate. That is, the cells can be grown three-dimensionally.
  • cell culture in which cells that do not adhere to the cell culture substrate can also be performed.
  • FIG. 1 is a photomicrograph of Example 1 one week after the start of culture.
  • FIG. 2 is a photomicrograph of Example 1 one month after the start of culture.
  • FIG. 3 is a photomicrograph of Example 1 two months after the start of culture.
  • FIG. 4 is a photomicrograph of Example 1 three months after the start of culture.
  • FIG. 5 is a photomicrograph of Example 3 two weeks after the start of culture.
  • FIG. 6 is a micrograph of Comparative Example 1 two weeks after the start of culture.
  • the cell culture substrate of the present invention comprises calcium phosphate and fibers. From the viewpoint of contribution to cell proliferation, it is preferable that calcium phosphate is exposed. More preferably, more than half of the surface of the substrate for cell culture is covered with calcium phosphate, which is preferably covered with calcium phosphate.
  • the material (raw material) of the fiber is not limited, and a general material can be used.
  • fiber materials include natural fiber (cotton, hemp, wool, etc.), regenerated fiber (rayon, cuvula), semi-synthetic fiber (acetate, promix), synthetic fiber (nylon, polyester, acrylic, vinylon, polychlorinated) Vinyl, vinylidene, polyolefin, polyurethane, polyclar, fluorocarbon, novoloid, PEEK, etc., inorganic fibers (alumina fibers, silicon carbide fibers and other ceramic fibers, glass fibers, carbon fibers, slag fibers, metal fibers, etc.) ).
  • the diameter of the fiber is not particularly limited, and may be submicron or several to several tens of microns. Moreover, the fiber which converged these ultrafine fibers may be used.
  • the fibers preferably form a non-woven fabric, but are not limited thereto.
  • nonwoven fabrics that are regularly woven, such as meshes fibers are irregularly entangled or in contact with each other, and have an appropriate space, thickness, and elasticity. Therefore, when cells adhere to each other at the part where the cells and the scaffold (cell culture substrate) are not in contact, the nonwoven fabric has a function of maintaining an appropriate distance between the cells and promotes the adhesion between the cells. Infer . It is particularly preferable that the fibers forming the nonwoven fabric are fixed to each other. Fibers stick together Therefore, even when the non-woven fabric is moved by exchanging the culture solution, the three-dimensional relationship of the fibers is difficult to change, and the cells generated in the gaps of the fibers are difficult to peel off. This is particularly important when performing long-term culture.
  • the nonwoven fabric to which the fibers are fixed is obtained, for example, by heating a nonwoven fabric made of a thermoplastic resin fiber or a fiber coated with a thermoplastic resin.
  • the fiber density of the nonwoven fabric is preferably relatively high from the viewpoint that the cell density (culture efficiency) can be increased.
  • a weight force per unit area of the nonwoven fabric of 0.1 to 0.3 g / 100 cm 2 is suitable for efficient cell culture, and more preferably 0.12 to 0.2 g / 100 cm 2 .
  • calcium phosphate promotes cell attachment to the fiber at the start of culture.
  • examples of calcium phosphate include primary calcium phosphate, secondary calcium phosphate, tertiary calcium phosphate and apatites (eg, hydroxyapatite, fluorapatite, hydrochloride apatite and carbonate apatite).
  • the method for containing the cell culture substrate S calcium phosphate is not particularly limited.
  • Examples of the method include a method in which the fiber to which calcium phosphate is adhered is heated to a temperature above the softening point so that the fiber is adhered to calcium phosphate, and a method in which the calcium phosphate is adhered to the fiber using a binder.
  • a slurry containing calcium phosphate is applied to the fiber, or the fiber is immersed in the slurry.
  • concentration of calcium phosphate slurry is preferably 0.1-70% by mass.
  • the particle size of calcium phosphate is preferably 10 nm to l 00 ⁇ 10 nrr! ⁇ 10 z m more preferred to be. Calcium phosphate with a particle size of less than 10 nm is difficult to synthesize.
  • Various dispersants can be added to the calcium phosphate slurry.
  • dispersants include polycarboxylic acid ammonium salts as anionic dispersants, polyimines and polyethyleneimines as cationic dispersants, TWEEN 20 as nonionic dispersants, and pyrroline as an inorganic ionic dispersant. Examples include sodium acid.
  • the calcium phosphate adheres to the fiber.
  • the fiber material for example, when the fiber surface is made of polyethylene, it is preferable to heat the fiber to 105-200 ° C. It is more preferable to heat to 110-150 ° C.
  • a method for adhering calcium phosphate to fibers by heat treatment at a temperature above the softening point is described in detail in Japanese Patent No. 2916068.
  • high-density culture refers to the density of cells (number of cells per unit volume) obtained by culturing by controlling physical, chemical, or biological culturing conditions, as compared to those under normal conditions. Say culture to enlarge.
  • high-density culture at what cell density depends on the type of cells to be cultured and the purpose of the culture, for example, 1 ⁇ 108 cells / mL, which is 100 times the density of static culture The above is sometimes called high-density culture.
  • 1 X 108 cells / mL is a guideline for high-density culture.
  • Examples of the culture apparatus of the present invention include a flask, a petri dish, and the like, which are generally used for culture and in which a cell culture substrate is placed.
  • One cell culture substrate may be placed in the container, or a plurality of (for example, 2 to 3) substrates may be stacked. When a plurality of cell culture substrates are stacked, there is an advantage that the cells can be cultured efficiently.
  • the culture instrument may include a medium, a culture reagent, a reagent or member for fixing the substrate, and the like.
  • cell culture substrate or culture apparatus of the present invention When the cell culture substrate or culture apparatus of the present invention is used, cell culture can be performed efficiently. For example, after a predetermined medium is put in a culture container, a cell culture substrate is added, and the substrate is acclimatized to the medium. Cell proliferation occurs when cells are entrained in a cell culture substrate that is compatible with the medium. A common medium can be used.
  • This cell culture method has the following advantages. (1) Human fibroblasts can be cultured by the cell culture method of the present invention, and most adherent cells such as stem cells can be cultured.
  • the cells can be grown even at positions not attached to the cell culture substrate, the cells can be cultured three-dimensionally. Therefore, high-density cultivation can be performed without complex or three-dimensional scaffolding.
  • Continuous culture can be performed for a long period of time while maintaining the proliferation of cells. That is, even without subculture, it can be continuously cultured for a long period of time by simply changing the medium periodically.
  • the cells proliferate three-dimensionally when the cells not in contact with the scaffold adhere to each other. For this reason, if the cells are continuously cultured, the cells can self-grow in the absence of a scaffold. Therefore, for example, when fibers are made of a material that decomposes and / or absorbs in vivo, there is a possibility that tissues and organs can be constructed by this cell culture method, and there is a possibility that it can be applied to human biomaterials for regenerative medicine. .
  • Hydroxyapatite particles (the primary particles with a particle size of 50 nm agglomerated into secondary particles with a particle size of 2 to 7 ⁇ m) are dispersed in water so that the solid concentration is 10% by mass, and the slurry is dispersed. Obtained.
  • a non-woven fabric made of polyester and having a surface coated with polyethylene was heated to fuse the fibers together. After this nonwoven fabric (average fiber diameter of about 10 ⁇ , 9cm x 8cm, sterilized) is immersed in a hydroxyapatite slurry, the nonwoven fabric is taken out of the slurry and heated with 130 ° C hot air to produce hydroxyapatite particles. Adhering to non-woven fabric I let you.
  • the obtained cell culture substrate was used as a scaffold, and cell culture was performed as shown below.
  • a cell culture substrate was added, and the culture medium was adapted to the cell culture substrate.
  • IX 106 normal human fibroblasts were placed in a cell culture substrate and cultured.
  • the medium used was a composition of FBS (final concentration 5%), antibiotics, L-ascorbic acid phosphate magnesium salt 0.2 mM, and the medium was changed at weekly intervals.
  • the cells Two months after the start of the culture, the cells further expanded into a membrane and proliferated, and the cells expanded in three dimensions throughout the thickness of the nonwoven fabric (see Fig. 3).
  • the cells spread in a membrane and covered the whole nonwoven fabric to form a three-dimensional cell mass (see Fig. 4).
  • Example 1 When the cell culture substrate of Example 1 is used as a scaffold, the cells adhere to each other even in a place where the cells and the cell culture substrate are not in contact with each other and spread into a film. It was able to grow originally.
  • Cell culture was performed using the same cell culture substrate as in Example 1, and the number of cells attached to the cell culture substrate and the flask and the amount of collagen produced were examined.
  • the trypsin treatment at 37 ° C for 1 hour was repeated 3 times.
  • the flask culture was performed in the same manner as in Example 1 except that the cell culture substrate was not added to the flask.
  • the number of adherent cells per unit area (lcm X lcm) of the flask in a 100% confluent state was determined.
  • the amount of collagen in the culture supernatant was measured.
  • the cell culture substrate of the present invention is suitable for cell culture and can be used for high-density culture.
  • Example 1 Comparison was made between the case where cell culture was performed using the same cell culture substrate as in Example 1 and the case where a non-woven fabric without Comparative Hydroxide (Comparative Example 1) was used.
  • Comparative Example 1 5 x 105 human normal fibroblasts were respectively inoculated into the cell culture substrate or Comparative Example 1, and the cells were cultured in the same manner as in Example 1 except that the medium was changed at intervals of 4 to 5 days. did.
  • FIG. 5 and FIG. 6 are photomicrographs showing cells 2 weeks after the start of culture.
  • Fig. 5 is a photomicrograph of the nonwoven fabric (Example 3) to which the hydroxyapatite is fixed
  • Fig. 6 is a photomicrograph of the non-woven fabric (Comparative Example 1) with the hydroxyapatite attached. .
  • the cell culture substrate when used, the cell culture substrate Cells were attached to many fibers (Fig. 5), whereas when non-woven fabric without hydroxyapatite was used, cells were attached only to a part of the fibers (Fig. 6).
  • Example 3 As a result of measuring the amount of collagen produced by the cells (the amount of collagen in the culture supernatant) one month after the start of culture, in Example 3, the amount of collagen produced was 5.6 x gZml (part other than the cell culture substrate) In the case of Comparative Example 1, the amount of collagen produced was 1.3 xg / ml (the amount of collagen produced by cells attached to parts other than the non-woven fabric). Was the value).
  • Example 3 From the comparison between Example 3 and Comparative Example 1, it was found that by adhering hydroxyapatite to the nonwoven fabric, cell attachment and proliferation were promoted and collagen production was increased.
  • the fiber density of the nonwoven fabric was examined.
  • the density of the nonwoven fabric fiber can be adjusted by the weight per unit area of the nonwoven fabric. Therefore, we prepared three types of nonwoven fabrics with different densities (weight per unit area).
  • Table 2 shows the weight per unit area (10 cm X IO cm) of each nonwoven fabric (number of grams of “nonwoven fabric only”).
  • a cell culture substrate (sample:! To 3) was obtained in the same manner as in Example 1 except that these non-woven fabrics were used, and Example 1 except that 4 x 10 5 human normal fibroblasts were infused into each sample. Culture was performed in the same manner as described above.
  • the column “nonwoven fabric + HAp” indicates the weight per unit area (10 cm ⁇ IO cm 2) of the cell culture substrate.
  • the column “HAp” indicates the weight of hydroxyapatite adhering to the nonwoven fabric.
  • the weight of the iodoxiapatite was determined by burning the cell culture substrate and measuring the weight of the residue.
  • the column of “nonwoven fabric only” indicates the weight per unit area of the nonwoven fabric.
  • the weight per unit area of the non-woven fabric was calculated by subtracting the value (A) force in the “non-woven fabric + HAp” column (A) and the value (B) in the “HAp” column.
  • the amount of collagen in Table 3 is the same as in Example 3 and Comparative Example 1, except for the amount of collagen produced by cells adhering to portions other than the non-woven fabric to which the cell culture substrate or hydroxyapatite is not attached. It is a value.
  • Cell culture was performed using one or more cell culture substrates. Place the culture medium in a 75 cm 2 flask, add one or more cell culture substrates, and infuse 5 ⁇ 105 human normal fibroblasts, and change the medium every 4 to 5 days. The culture was performed in the same manner as in Example 1 except that the above was performed.
  • Collagen content in the culture supernatant was measured 3 months after the start of the culture. The results are shown in Table 4 [Table 4] Number of cell culture substrates used Collagen content
  • Meviol Gel registered trademark, manufactured by Meviol Co., Ltd., hereinafter the same
  • Meviol gel is fluid Zonole-like under low-temperature conditions, and gel-like under conditions above the transition temperature (for example, culture conditions). After mixing under low-temperature conditions, cells are transferred to culture conditions. A culture substrate can be fixed.
  • a cell culture substrate was prepared in the same manner as in Example 1 except that the size of the nonwoven fabric was 3 cm X 7 cm. Put this cell culture substrate in a 10 cm diameter petri dish, put medium and meviol gel, fix the nonwoven, and infuse 2 X 105 human normal fibroblasts, and change the medium every 4-5 days Incubation was carried out in the same manner as in Example 1.
  • the culture was performed in the same manner as in Example 6 except that no mebiol gel was added, and was used as a control.
  • the amount of collagen was larger when the cell culture substrate was fixed to a petri dish.
  • the reasons for this are as follows: (a) If the cell culture substrate is not fixed to the petri dish, the cell culture substrate will float and / or move. It is conceivable that the amount of collagen produced by living cells is also reduced due to the effects of the dead cells.
  • a method of encapsulating cells after attaching them to a microcarrier was investigated. Place 2 X 106 human fibroblasts in a petri dish with a diameter of 10 cm, and add 0.5-2.0 g of microcarrier "CE LLYARD beads (Pentax Co., Ltd.)" to the next day. . During this time, more than half of the cells adhered to the microcarriers. Culturing was carried out in the same manner as in Example 1 except that the microcarriers to which the cells were attached were discharged onto the cell culture substrate instead of entraining the cells in the cell culture substrate.
  • Example 1 As a result, in Example 1, it took about 2 months for the collagen production to reach 20 ⁇ g / ml or more, whereas in this experiment, the same amount of collagen was produced in about 3 weeks. That's it.
  • This result shows that cell attachment and / or cell culture is more likely to occur when cells are attached to a microcarrier and then inoculated into a cell culture substrate, rather than after trypsinization and floating the cells as usual. Shows that growth can be accelerated.
  • a cell culture substrate was prepared in the same manner as in Example 1 except that the size of the nonwoven fabric was 3 cm ⁇ 7 cm.
  • a cell culture substrate and medium were placed in a flask having a bottom area of 25 cm 2 and cultured in the same manner as in Example 1 except that the cells involved were as shown in Table 6. Incubation continued for more than 3 months.
  • the cell culture substrate of the present invention can be applied to culture of almost all adherent cells.
  • the cell culture substrate of the present invention when used, there is an advantage that long-term continuous culture is possible.

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Abstract

A sheet-type cell culture substrate comprising calcium phosphate and fibers; a culture apparatus comprising the cell culture substrate; and a method for cell culture using the cell culture substrate or the culture apparatus.

Description

明 細 書  Specification
細胞培養用基材、培養用器具及び細胞培養方法  Cell culture substrate, culture instrument, and cell culture method
技術分野  Technical field
[0001] 本発明は、細胞を付着させて培養するのに用いられる基材、細胞培養用基材を備 えた培養用器具及び細胞培養方法に関する。  TECHNICAL FIELD [0001] The present invention relates to a substrate used for culturing with cells attached thereto, a culture instrument equipped with a cell culture substrate, and a cell culture method.
背景技術  Background art
[0002] 細胞を大別すると、浮遊系細胞と付着系細胞がある。浮遊細胞は生育や増殖に足 場を必要としない細胞であり、付着細胞は生育や増殖に足場を必要とする細胞であ る。再生医療で重要な幹細胞のほとんどは付着細胞であり、その培養には人為的に 作製した足場に基となる細胞を付着させることを要する。  [0002] Cells are roughly classified into suspension cells and adherent cells. Suspended cells are cells that do not require a scaffold for growth and proliferation, and adherent cells are cells that require a scaffold for growth and proliferation. Most stem cells important in regenerative medicine are adherent cells, and their culture requires attachment of cells based on artificially prepared scaffolds.
[0003] 付着細胞の足場として、ポリスチレン等の合成高分子、コラーゲン等の生物由来タ ンパク質等が利用されている。また足場となる基材に対する細胞接着性を大きくした り、足場となる基材にタンパク質が吸着し易くしたりするといつた改良も種々行われて いる。さらに、臨床応用の観点から、足場用の基材に PGA (ポリグリコール酸)等の生 体吸収性素材を用いる試みも行われている。これらの細胞培養用基材に細胞を付着 させて培養液に浸すと、基材を足場として細胞増殖が起こるので、基材表面に単層 の細胞層が形成する。しかし、再生医療が盛んになるにつれて、人工生体材料に求 められる要求はますます高まっており、足場となる基材に付着しない位置でも増殖し て、二層以上の細胞塊が生じるような細胞培養が望まれている。  [0003] As a scaffold for adherent cells, synthetic polymers such as polystyrene, biological proteins such as collagen, and the like are used. In addition, various improvements have been made when cell adhesion to a base material serving as a scaffold is increased or proteins are easily adsorbed to the base material serving as a scaffold. In addition, from the viewpoint of clinical application, attempts have been made to use bioabsorbable materials such as PGA (polyglycolic acid) as a scaffold base material. When cells are attached to these cell culture substrates and immersed in a culture solution, cell proliferation occurs using the substrate as a scaffold, so that a single cell layer is formed on the substrate surface. However, as regenerative medicine becomes popular, the demand for artificial biomaterials is increasing, and cells that proliferate at positions that do not adhere to the scaffolding base material and generate two or more cell clusters. Culture is desired.
[0004] 特許文献 1 (特開 2005-27532号）には微粒子と、親水性成分とを含む親水性高分 子ゲルからなる細胞培養基材が記載されている。この細胞培養基材を作製するには 、例えば微粒子、親水性成分のモノマー及び架橋剤を溶媒に均一に分散させた後、 モノマーを重合させる。微粒子としてはポリスチレンのような有機微粒子の他、シリカ、 ノ、イドロキシアパタイトのような無機微粒子も例示されてレ、る。特許文献 1に記載の細 胞培養基材に細胞を付着させると、親水性高分子ゲルを足場として細胞を増殖させ ることができる。しかし、この細胞培養基材を用いて培養をする場合、細胞の増殖は 親水性高分子ゲルに付着するように起こるだけである。 [0005] 特許文献 2 (特開 2001-157574号）には、生分解性ポリマーを含むハニカム構造体 からなる細胞培養用基材が記載されている。ハニカム構造体を作製するには、生分 解性ポリマー及び両親媒性ポリマーと、疎水性有機溶媒とを含む溶液を相対湿度 50 〜95%の大気中で基板上にキャストし、疎水性有機溶媒を徐々に蒸散させると同時 にキャスト液表面で結露させ、結露により生じた微小水滴を蒸発させる。この細胞培 養用基材を用いて細胞培養する場合も、二次元的な細胞増殖しか起こらず、単層の 細胞しか生じなレ、。またこのハニカム構造体の作製には疎水性有機溶媒が必須であ り、安全性の観点でも問題があると思われる。 [0004] Patent Document 1 (Japanese Patent Application Laid-Open No. 2005-27532) describes a cell culture substrate comprising a hydrophilic polymer gel containing fine particles and a hydrophilic component. In order to prepare this cell culture substrate, for example, fine particles, a monomer of a hydrophilic component, and a crosslinking agent are uniformly dispersed in a solvent, and then the monomer is polymerized. Examples of the fine particles include organic fine particles such as polystyrene, and inorganic fine particles such as silica, silica and idoxyapatite. When cells are attached to the cell culture substrate described in Patent Document 1, the cells can be proliferated using the hydrophilic polymer gel as a scaffold. However, when culturing using this cell culture substrate, cell growth only occurs to adhere to the hydrophilic polymer gel. [0005] Patent Document 2 (Japanese Patent Laid-Open No. 2001-157574) describes a cell culture substrate comprising a honeycomb structure containing a biodegradable polymer. In order to fabricate a honeycomb structure, a solution containing a biodegradable polymer and an amphiphilic polymer and a hydrophobic organic solvent is cast on a substrate in an atmosphere having a relative humidity of 50 to 95%, and the hydrophobic organic solvent is obtained. As the water evaporates gradually, condensation occurs on the surface of the casting liquid, and the water droplets generated by the condensation evaporate. When cells are cultured using this cell culture substrate, only two-dimensional cell growth occurs, and only single-layer cells are generated. In addition, a hydrophobic organic solvent is indispensable for the production of this honeycomb structure, and there seems to be a problem from the viewpoint of safety.
[0006] 特許文献 3 (特開 2005-52224号）には、連通孔を有する生体吸収性有機多孔体と、 骨親和性無機材料とが複合化された骨再生用基材が記載されている。例えば加圧、 減圧等の手段を用い、乳酸- ε -カプロラ外ン共重合等からなる生体吸収性有機多 孔体の連通孔にハイドロキシアパタイト等の骨親和性無機材料を充填すると、この骨 再生用基材を得ることができる。多孔体の形態としては繊維、織地シート、不織布等 が挙げられている。生体内に埋入されると、生体吸収性材料は溶け出すので、骨親 和性無機材料は単に生体吸収性有機多孔体の連通孔内に充填されてレ、るだけでも 骨形成に寄与しうる。しかし、本発明者らによる細胞培養に関する研究の結果、骨親 和性無機材料が細胞増殖に寄与するには、生体吸収性有機多孔体の連通孔に充 填されているだけでは不十分であることが分かった。さらに、連通孔に骨親和性無機 材料が充填された基材は、細胞が増殖するための適度な空間を有していないため、 培養には適してレ、なレ、ことも分かった。  [0006] Patent Document 3 (Japanese Patent Laid-Open No. 2005-52224) describes a bone regeneration base material in which a bioabsorbable organic porous material having communication holes and a bone-compatible inorganic material are combined. . For example, by using means such as pressurization, decompression, etc., when the bioresorbable organic porous material composed of lactic acid-ε-caprolain copolymer is filled with a bone-compatible inorganic material such as hydroxyapatite, this bone regeneration A substrate for use can be obtained. Examples of the form of the porous body include fibers, woven sheets, and non-woven fabrics. Since the bioabsorbable material melts when implanted in the living body, the bone-friendly inorganic material is simply filled in the communicating holes of the bioabsorbable organic porous material and contributes to bone formation. sell. However, as a result of studies on cell culture by the present inventors, it is not sufficient that the bone-friendly inorganic material is filled in the communicating holes of the bioabsorbable organic porous material in order to contribute to cell growth. I understood that. Furthermore, it was also found that the base material in which the communicating holes are filled with the bone-compatible inorganic material does not have an appropriate space for the cells to grow, so that it is suitable for culture.
[0007] なお、従来の細胞培養は、高密度培養も含め、各細胞が足場に付着する必要があ るので、基本的に単層培養である。そのため、細胞を三次元的に増殖させることが難 しい。細胞を三次元的に増殖させうる基材は、特に切望されている。  [0007] Conventional cell culture, including high-density culture, is basically a monolayer culture because each cell needs to adhere to the scaffold. Therefore, it is difficult to grow the cells three-dimensionally. Substrates that can grow cells three-dimensionally are particularly desired.
[0008] 特許文献 1：特開 2005-27532号公報  [0008] Patent Document 1: Japanese Patent Application Laid-Open No. 2005-27532
特許文献 2：特開 2001-157574号公報  Patent Document 2: Japanese Patent Laid-Open No. 2001-157574
特許文献 3：特開 2005-52224号公報  Patent Document 3: Japanese Patent Laid-Open No. 2005-52224
発明の開示  Disclosure of the invention
発明が解決しょうとする課題 [0009] 本発明の目的は、基材に付着しない細胞も生じさせ得る細胞培養用手段及びかか る細胞培養用手段を用いた細胞培養方法を提供することである。 Problems to be solved by the invention [0009] An object of the present invention is to provide a cell culturing means capable of generating cells that do not adhere to a substrate and a cell culturing method using such a cell culturing means.
課題を解決するための手段  Means for solving the problem
[0010] 上記課題に鑑み鋭意研究の結果、本発明者らは、リン酸カルシウム及び繊維から なるシートを細胞培養に用いると、シートの表面に細胞が増殖するば力りでなぐシー トに直接付着しない細胞も生じさせ得ることを発見し、本発明に想到した。  [0010] As a result of diligent research in view of the above problems, the present inventors, when using a sheet made of calcium phosphate and fiber for cell culture, do not directly adhere to the sheet that is stretched on the surface of the sheet by the force of the cell. It was discovered that cells could also be generated and the present invention was conceived.
[0011] すなわち、本発明の細胞培養用基材は、シート状であって、リン酸カルシウム及び 繊維からなることを特徴とする。  [0011] That is, the cell culture substrate of the present invention is sheet-shaped and is characterized by comprising calcium phosphate and fibers.
[0012] 細胞培養用基材はリン酸カルシウムによって覆われているのが好ましい。前記繊維 は不織布を形成しているのが好ましぐ不織布の単位面積あたりの重量は 0.1〜0.3 g /100 cm2であるのが好ましレ、。前記リン酸カルシウムはハイドロキシアパタイトである のが好ましい。この細胞培養用基材を用いて細胞培養を行うと、はじめは細胞培養 用基材を足場として細胞が増殖するが、次第に足場から離れた位置でも、細胞同士 が接着して細胞増殖が起こる。そのため、最終的には、細胞培養用基材全体を覆う ように細胞塊を形成させることができる。このような細胞培養を行うことができる本発明 の細胞培養用基材は、三次元培養に好適である。また高密度培養、連続長期培養 にも使用可能である。 [0012] The cell culture substrate is preferably covered with calcium phosphate. The fiber preferably forms a nonwoven fabric, and the weight per unit area of the nonwoven fabric is preferably 0.1 to 0.3 g / 100 cm 2 . The calcium phosphate is preferably hydroxyapatite. When cell culture is performed using this cell culture substrate, the cells initially grow using the cell culture substrate as a scaffold, but gradually cells adhere to each other even at a position away from the scaffold, and cell proliferation occurs. Therefore, finally, a cell mass can be formed so as to cover the entire cell culture substrate. The cell culture substrate of the present invention capable of performing such cell culture is suitable for three-dimensional culture. It can also be used for high-density culture and continuous long-term culture.
[0013] 本発明の細胞培養器具は、本発明の細胞培養用基材を具備することを特徴とする  [0013] A cell culture instrument of the present invention comprises the cell culture substrate of the present invention.
[0014] 本発明の細胞培養方法は、本発明の細胞培養用基材又は本発明の培養用器具 を用いることを特徴とする。本発明の細胞培養用基材を用いることにより、細胞同士 が接着することで、細胞と細胞培養用基材が接していない部分でも細胞を増殖させ ることができる。つまり、細胞を三次元的に増殖させることができる。 [0014] The cell culture method of the present invention is characterized by using the cell culture substrate of the present invention or the culture instrument of the present invention. By using the cell culture substrate of the present invention, the cells adhere to each other, so that the cells can be proliferated even in a portion where the cells are not in contact with the cell culture substrate. That is, the cells can be grown three-dimensionally.
発明の効果  The invention's effect
[0015] 本発明の細胞培養用基材を用いることにより、細胞培養用基材に付着しない細胞 も生じる細胞培養を行うことができる。  [0015] By using the cell culture substrate of the present invention, cell culture in which cells that do not adhere to the cell culture substrate can also be performed.
図面の簡単な説明  Brief Description of Drawings
[0016] [図 1]培養開始から 1週間後の実施例 1の顕微鏡写真である。 [図 2]培養開始から 1力月後の実施例 1の顕微鏡写真である。 FIG. 1 is a photomicrograph of Example 1 one week after the start of culture. FIG. 2 is a photomicrograph of Example 1 one month after the start of culture.
[図 3]培養開始から 2力月後の実施例 1の顕微鏡写真である。  FIG. 3 is a photomicrograph of Example 1 two months after the start of culture.
[図 4]培養開始から 3力月後の実施例 1の顕微鏡写真である。  FIG. 4 is a photomicrograph of Example 1 three months after the start of culture.
[図 5]培養開始から 2週間後の実施例 3の顕微鏡写真である。  FIG. 5 is a photomicrograph of Example 3 two weeks after the start of culture.
[図 6]培養開始から 2週間後の比較例 1の顕微鏡写真である。  FIG. 6 is a micrograph of Comparative Example 1 two weeks after the start of culture.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0017] [1]細胞培養用基材  [1] Cell culture substrate
本発明の細胞培養用基材は、リン酸カルシウム及び繊維からなる。細胞増殖への 寄与の観点で、リン酸カルシウムが露出しているのが好ましい。細胞培養用基材の表 面の 1/3以上がリン酸カルシウムによって覆われているのが好ましぐ半分以上が覆 われているのがより好ましい。  The cell culture substrate of the present invention comprises calcium phosphate and fibers. From the viewpoint of contribution to cell proliferation, it is preferable that calcium phosphate is exposed. More preferably, more than half of the surface of the substrate for cell culture is covered with calcium phosphate, which is preferably covered with calcium phosphate.
[0018] リン酸カルシウムを含有したり、付着させたりすることができる限り、繊維の材質 (原 料）は限定されず、一般的なものを用いることができる。繊維の材質として、例えば天 然繊維 (木綿、麻、羊毛等）、再生繊維（レーヨン、キュブラ）、半合成繊維 (アセテート 、プロミックス）、合成繊維（ナイロン、ポリエステル、アクリル系、ビニロン、ポリ塩化ビ ニル、ビニリデン、ポリオレフイン系、ポリウレタン、ポリクラール、フルォロカーボン系、 ノボロイド系、 PEEK等）、無機繊維（アルミナ繊維、シリコンカーバイド繊維等のセラミ ック繊維、ガラス繊維、炭素繊維、スラグ繊維、金属繊維等）が挙げられる。また生体 内で分解及び Z又は吸収する素材、例えば PGA (ポリグリコール酸）、 PLGA (ポリ乳 酸とポリダリコール酸の共重合体)等からなっても良レ、。繊維の径も特に限定されず、 サブミクロンでも良いし、数マイクロ〜数 10マイクロでも良い。またこれらの極細繊維を 収束した繊維でも良い。  [0018] As long as calcium phosphate can be contained or adhered, the material (raw material) of the fiber is not limited, and a general material can be used. Examples of fiber materials include natural fiber (cotton, hemp, wool, etc.), regenerated fiber (rayon, cuvula), semi-synthetic fiber (acetate, promix), synthetic fiber (nylon, polyester, acrylic, vinylon, polychlorinated) Vinyl, vinylidene, polyolefin, polyurethane, polyclar, fluorocarbon, novoloid, PEEK, etc., inorganic fibers (alumina fibers, silicon carbide fibers and other ceramic fibers, glass fibers, carbon fibers, slag fibers, metal fibers, etc.) ). It can also be made of a material that decomposes and absorbs or absorbs in vivo, such as PGA (polyglycolic acid), PLGA (copolymer of polylactic acid and polydaricholic acid). The diameter of the fiber is not particularly limited, and may be submicron or several to several tens of microns. Moreover, the fiber which converged these ultrafine fibers may be used.
[0019] 繊維は、不織布を形成しているのが好ましいが、それに限定されない。  [0019] The fibers preferably form a non-woven fabric, but are not limited thereto.
不織布は、メッシュ等規則的に織られたものと異なり、繊維同士が不規則に絡まつ たり接触したりしており、適度な空間、厚さ及び弾力を有している。そのため、細胞と 足場 (細胞培養用基材）が接していない部分で細胞同士が接着する際、不織布がそ の細胞間の適度な距離を保つ機能を持ち、細胞同士の接着を促していると推測する 。不織布を形成する繊維同士は固着されているのが特に好ましい。繊維同士が固着 されていることで、培養液を交換したりして不織布が動かされる場合にも繊維の立体 的な関係が変わり難 繊維の隙間に生じた細胞が剥がれたりし難い。このことは長 期培養を行う場合には、特に重要である。繊維同士が固着された不織布は、例えば 熱可塑性樹脂性の繊維や熱可塑性樹脂でコートされた繊維からなる不織布を加熱 することにより得られる。 Unlike nonwoven fabrics that are regularly woven, such as meshes, fibers are irregularly entangled or in contact with each other, and have an appropriate space, thickness, and elasticity. Therefore, when cells adhere to each other at the part where the cells and the scaffold (cell culture substrate) are not in contact, the nonwoven fabric has a function of maintaining an appropriate distance between the cells and promotes the adhesion between the cells. Infer . It is particularly preferable that the fibers forming the nonwoven fabric are fixed to each other. Fibers stick together Therefore, even when the non-woven fabric is moved by exchanging the culture solution, the three-dimensional relationship of the fibers is difficult to change, and the cells generated in the gaps of the fibers are difficult to peel off. This is particularly important when performing long-term culture. The nonwoven fabric to which the fibers are fixed is obtained, for example, by heating a nonwoven fabric made of a thermoplastic resin fiber or a fiber coated with a thermoplastic resin.
[0020] 不織布の繊維密度は、細胞密度 (培養効率)を高くできる点で、比較的高い方が好 ましい。不織布の単位面積あたりの重量力 0.1〜0.3 g/100 cm2であると、効率的な 細胞培養に好適であり、 0.12〜0.2 g/100 cm2であるのがより好ましい。 [0020] The fiber density of the nonwoven fabric is preferably relatively high from the viewpoint that the cell density (culture efficiency) can be increased. A weight force per unit area of the nonwoven fabric of 0.1 to 0.3 g / 100 cm 2 is suitable for efficient cell culture, and more preferably 0.12 to 0.2 g / 100 cm 2 .
[0021] リン酸カルシウムは、培養開始時に繊維に細胞が付着するのを促進すると推測する 。リン酸カルシウムの例として第 1リン酸カルシウム、第 2リン酸カルシウム、第 3リン酸 カルシウム及びアパタイト類（例えばハイドロキシアパタイト、フッ素アパタイト、塩酸ァ パタイト及び炭酸アパタイト）が挙げられる。  [0021] It is assumed that calcium phosphate promotes cell attachment to the fiber at the start of culture. Examples of calcium phosphate include primary calcium phosphate, secondary calcium phosphate, tertiary calcium phosphate and apatites (eg, hydroxyapatite, fluorapatite, hydrochloride apatite and carbonate apatite).
[0022] 細胞培養用基材カ Sリン酸カルシウムを含有するようにする方法は、特に限定されな レ、。その方法として、例えばリン酸カルシウムを付着させた繊維を軟化点以上に加熱 することにより、繊維をリン酸カルシウムに接着させる方法や、バインダーを用いてリン 酸カルシウムを繊維に接着する方法が挙げられる。  [0022] The method for containing the cell culture substrate S calcium phosphate is not particularly limited. Examples of the method include a method in which the fiber to which calcium phosphate is adhered is heated to a temperature above the softening point so that the fiber is adhered to calcium phosphate, and a method in which the calcium phosphate is adhered to the fiber using a binder.
[0023] リン酸カルシウムを繊維に付着させるには、リン酸カルシウムを含有するスラリーを 繊維に塗布したり、スラリーに繊維を浸漬させたりすれば良レ、。リン酸カルシウムスラリ 一の濃度は 0.1〜70質量％とするのが好ましレ、。リン酸カルシウムの粒径は 10 nm〜l 00 μ πιであるのが好ましぐ 10 nrr！〜 10 z mであるのがより好ましレ、。粒径 10 nm未満の リン酸カルシウムは合成し難ぐ粒径 100 z m超の粒子は基材から欠落し易過ぎる。リ ン酸カルシウムスラリーには各種の分散剤を添加しても良レ、。分散剤の例としてァニ オン系分散剤であるポリカルボン酸アンモニゥム塩、カチオン系分散剤であるポリイミ ン及びポリエチレンィミン、非イオン系分散剤である TWEEN20、並びに無機イオン系 分散剤であるピロリン酸ナトリウムが挙げられる。  [0023] In order to attach calcium phosphate to the fiber, a slurry containing calcium phosphate is applied to the fiber, or the fiber is immersed in the slurry. The concentration of calcium phosphate slurry is preferably 0.1-70% by mass. The particle size of calcium phosphate is preferably 10 nm to l 00 μπι 10 nrr! ~ 10 z m more preferred to be. Calcium phosphate with a particle size of less than 10 nm is difficult to synthesize. Various dispersants can be added to the calcium phosphate slurry. Examples of dispersants include polycarboxylic acid ammonium salts as anionic dispersants, polyimines and polyethyleneimines as cationic dispersants, TWEEN 20 as nonionic dispersants, and pyrroline as an inorganic ionic dispersant. Examples include sodium acid.
[0024] リン酸カルシウムスラリーに浸漬させた後で、繊維をスラリーから取り出して軟ィ匕点 以上の温度にすると、リン酸カルシウムは繊維に固着する。繊維の材質にもよるが、 例えば繊維表面がポリエチレンからなる場合、繊維を 105〜200°Cに加熱するのが好 ましぐ 110〜150°Cに加熱するのがより好ましレ、。軟化点以上の温度で熱処理するこ とによって、リン酸カルシウムを繊維に接着させる方法は、特許第 2916068号に詳細 に記載されている。なお再生医療等に用いる細胞を培養する場合や、人体等に適用 するコラーゲン等を産生する場合、バインダーを用いない方法の方が、健康保持等 の観点から好ましい。 [0024] After the fiber is taken out of the slurry after being immersed in the calcium phosphate slurry and brought to a temperature equal to or higher than the soft saddle point, the calcium phosphate adheres to the fiber. Depending on the fiber material, for example, when the fiber surface is made of polyethylene, it is preferable to heat the fiber to 105-200 ° C. It is more preferable to heat to 110-150 ° C. A method for adhering calcium phosphate to fibers by heat treatment at a temperature above the softening point is described in detail in Japanese Patent No. 2916068. When cells used for regenerative medicine or the like are cultured, or when collagen or the like to be applied to the human body or the like is produced, a method using no binder is preferable from the viewpoint of maintaining health.
[0025] [2]培養用器具 [0025] [2] Culture equipment
本発明の細胞培養用基材を用いた細胞培養においては、細胞培養用基材表面に 細胞が増殖するばかりでなぐ足場に接しない細胞培養も生じるので、細胞を三次元 的に増殖させること力 Sできる。このような細胞培養用基材を具備する培養用器具は、 高密度培養に使用可能である。本明細書中、高密度培養とは、物理的、化学的又は 生物的な培養の条件をコントロールすることで培養により得られる細胞の密度（単位 体積あたりの細胞数）を通常の条件によるものより大きくする培養を言う。従って、細 胞密度がどの程度の場合に高密度培養と呼ぶかは、培養する細胞の種類や培養の 目的によっては異なるが、例えば静置培養の密度の 100倍である 1 X 108個/ mL以 上であると、高密度培養と呼ばれる場合がある。 1 X 108個/ mLは高密度培養の目 安と言える。  In the cell culture using the cell culture substrate of the present invention, there is a cell culture not only in contact with the scaffold but also growing on the surface of the cell culture substrate. S can. A culture instrument comprising such a cell culture substrate can be used for high-density culture. In this specification, high-density culture refers to the density of cells (number of cells per unit volume) obtained by culturing by controlling physical, chemical, or biological culturing conditions, as compared to those under normal conditions. Say culture to enlarge. Therefore, what is called high-density culture at what cell density depends on the type of cells to be cultured and the purpose of the culture, for example, 1 × 108 cells / mL, which is 100 times the density of static culture The above is sometimes called high-density culture. 1 X 108 cells / mL is a guideline for high-density culture.
[0026] 本発明の培養用器具の例として、フラスコ、シャーレ等、培養に一般に用いられて レ、る容器に、細胞培養用基材を入れたものが挙げられる。容器に入れられる細胞培 養用基材は 1枚でもよいし、複数枚 (例えば 2〜3枚)重ねられてもよい。細胞培養用 基材が複数枚重ねられていると、効率よく培養できるという利点がある。培養用器具 は細胞培養用基材の他に培地、培養用試薬、前記基材を固定する試薬又は部材等 を含んでもよい。  [0026] Examples of the culture apparatus of the present invention include a flask, a petri dish, and the like, which are generally used for culture and in which a cell culture substrate is placed. One cell culture substrate may be placed in the container, or a plurality of (for example, 2 to 3) substrates may be stacked. When a plurality of cell culture substrates are stacked, there is an advantage that the cells can be cultured efficiently. In addition to the cell culture substrate, the culture instrument may include a medium, a culture reagent, a reagent or member for fixing the substrate, and the like.
[0027] [3]細胞培養方法  [0027] [3] Cell culture method
本発明の細胞培養用基材又は培養用器具を用いると、効率よく細胞培養を行うこと ができる。例えば培養容器に所定の培地を入れた後、細胞培養用基材を入れ、その 基材を培地に馴染ませる。培地に馴染んだ細胞培養用基材に細胞をまき込むと、細 胞増殖が起こる。培地は、一般的なのものを用いることができる。  When the cell culture substrate or culture apparatus of the present invention is used, cell culture can be performed efficiently. For example, after a predetermined medium is put in a culture container, a cell culture substrate is added, and the substrate is acclimatized to the medium. Cell proliferation occurs when cells are entrained in a cell culture substrate that is compatible with the medium. A common medium can be used.
[0028] この細胞培養方法には、以下のような有利性がある。 (1)本発明の細胞培養方法により、ヒト繊維芽細胞を培養可能であるほか、幹細胞等 ほとんどの付着細胞を培養可能である。 [0028] This cell culture method has the following advantages. (1) Human fibroblasts can be cultured by the cell culture method of the present invention, and most adherent cells such as stem cells can be cultured.
(2)細胞培養用基材に付着しない位置にも細胞を増殖させることができるので、三次 元的に細胞培養できる。よって足場を複雑又は立体的に構成しなくても、高密度培 養ができる。  (2) Since the cells can be grown even at positions not attached to the cell culture substrate, the cells can be cultured three-dimensionally. Therefore, high-density cultivation can be performed without complex or three-dimensional scaffolding.
(3)細胞の増殖性を維持した状態で、長期間連続培養を行うことができる。即ち、継 代培養をしなくても、定期的に培地交換するだけで、長期間連続して培養を行うこと ができる。  (3) Continuous culture can be performed for a long period of time while maintaining the proliferation of cells. That is, even without subculture, it can be continuously cultured for a long period of time by simply changing the medium periodically.
(4)足場と接触していない細胞同士が接着することにより、細胞が三次元的に増殖す る。そのため連続して培養を行うと、足場がない状態で細胞が自己増殖できる状態に なる。従って、例えば繊維が生体内で分解及び/又は吸収する素材からなる場合、 この細胞培養方法によって組織や器官を構築できる可能性があり、再生医療用の人 ェ生体材料に適用できる可能性がある。  (4) The cells proliferate three-dimensionally when the cells not in contact with the scaffold adhere to each other. For this reason, if the cells are continuously cultured, the cells can self-grow in the absence of a scaffold. Therefore, for example, when fibers are made of a material that decomposes and / or absorbs in vivo, there is a possibility that tissues and organs can be constructed by this cell culture method, and there is a possibility that it can be applied to human biomaterials for regenerative medicine. .
[0029] なおこの細胞培養方法において、長期間連続培養が可能な理由は、 7火のようなメ 力二ズムに基づくと推測する。本発明の細胞培養用基材を用いて細胞培養を行うと、 細胞と足場が接触していない部分で細胞同士が接着し、生体内における細胞間間 隙と同様の構造を構築する。それにより、個々の細胞が直接培地を用いて代謝を行 レ、、必要な物質を自ら合成できるようになり、壊死を回避する。  [0029] It is assumed that the reason why long-term continuous culture is possible in this cell culturing method is based on a mechanism such as 7 fires. When cell culture is performed using the cell culture substrate of the present invention, the cells adhere to each other at a portion where the cells and the scaffold are not in contact with each other, and a structure similar to the intercellular space in the living body is constructed. As a result, individual cells can directly metabolize using the medium and synthesize necessary substances themselves, thereby avoiding necrosis.
実施例  Example
[0030] 本発明を以下の実施例によってさらに詳細に説明するが、本発明はそれらに限定 されるものではない。  [0030] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.
[0031] 実施例 1 [0031] Example 1
ハイドロキシアパタイト粒子（粒径 50 nmの一次粒子が凝集して粒径 2〜7 μ mの二 次粒子になったもの）を固形分濃度が 10質量％になるように水に分散させ、スラリー を得た。ポリエステルからなり、表面にポリエチレンがコートされた繊維からなる不織 布を加熱し、繊維同士を融着した。この不織布（平均繊維径約 10 μ πι、 9cm X 8cm, 滅菌済み）をハイドロキシアパタイトスラリーに浸漬させた後、不織布をスラリーから取 り出し、 130°Cの熱風を当てることにより、ハイドロキシアパタイト粒子を不織布に固着 させた。得られた細胞培養用基材を足場として用レ、、次に示すように細胞培養を行つ た。底面積 75 cm2のフラスコに培地を入れた後、細胞培養用基材を入れ、細胞培養 用基材に培地を馴染ませた。その後、細胞培養用基材に I X 106個のヒト正常線維 芽細胞をまき込み、培養を行った。培地には FBS (最終濃度 5%)、抗生物質、 L-ァス コルビン酸リン酸エステルマグネシウム塩 0.2 mMの組成のものを用い、 1週間間隔で 培地交換を行った。 Hydroxyapatite particles (the primary particles with a particle size of 50 nm agglomerated into secondary particles with a particle size of 2 to 7 μm) are dispersed in water so that the solid concentration is 10% by mass, and the slurry is dispersed. Obtained. A non-woven fabric made of polyester and having a surface coated with polyethylene was heated to fuse the fibers together. After this nonwoven fabric (average fiber diameter of about 10 μπι, 9cm x 8cm, sterilized) is immersed in a hydroxyapatite slurry, the nonwoven fabric is taken out of the slurry and heated with 130 ° C hot air to produce hydroxyapatite particles. Adhering to non-woven fabric I let you. The obtained cell culture substrate was used as a scaffold, and cell culture was performed as shown below. After the culture medium was put into a flask having a bottom area of 75 cm 2 , a cell culture substrate was added, and the culture medium was adapted to the cell culture substrate. Thereafter, IX 106 normal human fibroblasts were placed in a cell culture substrate and cultured. The medium used was a composition of FBS (final concentration 5%), antibiotics, L-ascorbic acid phosphate magnesium salt 0.2 mM, and the medium was changed at weekly intervals.
[0032] 結果は次の通りである。 [0032] The results are as follows.
培養開始当初、細胞は細胞培養用基材とフラスコ底面の両方に付着したが、付着 した細胞の絶対量は細胞培養用基材よりフラスコ底面の方が多かった。これは、フラ スコ底面積の方が細胞培養用基材の面積より大きいためと推測する。  At the beginning of the culture, cells adhered to both the cell culture substrate and the bottom of the flask, but the absolute amount of attached cells was greater on the flask bottom than the cell culture substrate. This is presumably because the area of the flask bottom is larger than the area of the cell culture substrate.
培養開始から 1週間後、細胞培養用基材の繊維に細胞が絡みつくように付着し、増 殖してレ、ることを確認した（図 1参照）。  One week after the start of the culture, it was confirmed that the cells attached to the fibers of the cell culture substrate so as to be entangled, and grew and re-applied (see Fig. 1).
培養開始から 1力月後、足場となる繊維がない部分でも細胞同士が接着し、繊維と 繊維の間に細胞が膜状に拡がって増殖していることを確認した（図 2参照）。  One month after the start of the culture, it was confirmed that cells adhered to each other even in the part where there was no fiber as a scaffold, and the cells spread and proliferated between the fibers (see Fig. 2).
培養開始から 2力月後、細胞がさらに膜状に拡がって増殖し、細胞が不織布の厚さ の全体に立体的に拡がった（図 3参照）。  Two months after the start of the culture, the cells further expanded into a membrane and proliferated, and the cells expanded in three dimensions throughout the thickness of the nonwoven fabric (see Fig. 3).
培養開始から 3力月後、細胞が膜状に拡がって不織布の全体を覆い、三次元的な 細胞塊を形成した（図 4参照）。  Three months after the start of culture, the cells spread in a membrane and covered the whole nonwoven fabric to form a three-dimensional cell mass (see Fig. 4).
[0033] 実施例 1の細胞培養用基材を足場として用いた場合、細胞と細胞培養用基材が全 く接していない場所でも、細胞同士が接着して膜状に拡がるため、細胞を三次元的 に増殖させることができた。 [0033] When the cell culture substrate of Example 1 is used as a scaffold, the cells adhere to each other even in a place where the cells and the cell culture substrate are not in contact with each other and spread into a film. It was able to grow originally.
[0034] 実施例 2 [0034] Example 2
実施例 1と同じ細胞培養用基材を用いて細胞培養を行い、細胞培養用基材及びフ ラスコに付着した細胞数とコラーゲン産生量を調べた。  Cell culture was performed using the same cell culture substrate as in Example 1, and the number of cells attached to the cell culture substrate and the flask and the amount of collagen produced were examined.
[0035] 実施例 1と同様にして培養を行った後、 100。/0コンフルェントの状態における細胞培 養用基材の不織布の単位面積（lcm X 1cm)あたりの付着細胞数と、培養上清中のコ ラーゲン量を測定した。細胞培養用基材のサイズは 9cm X 8cmであったので、面積 は 72 cm2であった。付着細胞数の計測に当たっては、トリプシン処理により細胞を浮 遊させた後、細胞数をカウントした。なお不織布と細胞との結合は強固であったため[0035] After culturing in the same manner as in Example 1, 100. The number of adherent cells per unit area (lcm × 1 cm) of the nonwoven fabric of the cell culture substrate in the state of / 0 confluent and the amount of collagen in the culture supernatant were measured. Since the size of the cell culture substrate was 9 cm × 8 cm, the area was 72 cm 2 . When measuring the number of adherent cells, the cells are floated by trypsin treatment. After allowing to migrate, the number of cells was counted. Because the bond between the nonwoven fabric and the cells was strong
、 37°C、 1時間のトリプシン処理を 3回繰り返して行った。 The trypsin treatment at 37 ° C for 1 hour was repeated 3 times.
また対照として、フラスコに細胞培養用基材を入れない以外実施例 1と同様にして フラスコ培養を行レ、、 100%コンフルェントの状態におけるフラスコの単位面積（lcm X lcm)あたりの付着細胞数と、培養上清中のコラーゲン量を測定した。  As a control, the flask culture was performed in the same manner as in Example 1 except that the cell culture substrate was not added to the flask. The number of adherent cells per unit area (lcm X lcm) of the flask in a 100% confluent state was determined. The amount of collagen in the culture supernatant was measured.
[0036] 結果を表 1に示す。なお細胞培養時には、細胞培養用基材だけでなぐフラスコ底 面にも細胞が付着し、増殖する。そこで「実施例 1」から対照である「フラスコ培養」を Iレ、た値を、細胞培養用基材で増殖した細胞に由来するコラーゲン量と推定する。 The results are shown in Table 1. During cell culture, cells adhere to and grow on the bottom of the flask, which is made of only the cell culture substrate. Therefore, the value obtained from “flask culture” as a control from “Example 1” is estimated as the amount of collagen derived from cells grown on the cell culture substrate.
1]  1]
Figure imgf000011_0001
Figure imgf000011_0001
[0037] 表 1に示す通り、細胞培養用基材を用いて培養を行う場合、フラスコ培養（単層培 養）の場合と比較して、付着細胞数が 5.3倍、コラーゲン量が 4.0倍多かった。これは、 培養細胞が細胞培養用基材に好適に付着でき、かつ立体的に増殖できたためであ ると推測する。  [0037] As shown in Table 1, when culturing using a cell culture substrate, the number of adherent cells is 5.3 times and the amount of collagen is 4.0 times greater than in the case of flask culture (monolayer culture). It was. This is presumed to be because the cultured cells were able to adhere suitably to the cell culture substrate and were able to multiply three-dimensionally.
実施例 2の培養結果から、本発明の細胞培養用基材は細胞培養に適している上、 高密度培養に使用可能であることが分かった。  From the culture results of Example 2, it was found that the cell culture substrate of the present invention is suitable for cell culture and can be used for high-density culture.
[0038] 実施例 3及び比較例 1 [0038] Example 3 and Comparative Example 1
実施例 1と同じ細胞培養用基材を用いて細胞培養した場合と、ハイドロキシァパタ イトを付着させていない不織布（比較例 1)を用いた場合とを比較した。本実験では、 ヒト正常線維芽細胞を細胞培養用基材又は比較例 1にそれぞれ 5 X 105個まき込み、 培地交換を 4〜5日間隔で行った以外実施例 1と同様にして細胞を培養した。  Comparison was made between the case where cell culture was performed using the same cell culture substrate as in Example 1 and the case where a non-woven fabric without Comparative Hydroxide (Comparative Example 1) was used. In this experiment, 5 x 105 human normal fibroblasts were respectively inoculated into the cell culture substrate or Comparative Example 1, and the cells were cultured in the same manner as in Example 1 except that the medium was changed at intervals of 4 to 5 days. did.
[0039] 図 5及び図 6は、培養開始から 2週間後の細胞を示す顕微鏡写真である。図 5はハ イドロキシアパタイトを固着させた不織布（実施例 3)の顕微鏡写真であり、図 6はハイ ドロキシアパタイトを付着させてレ、なレ、不織布（比較例 1)の顕微鏡写真である。 FIG. 5 and FIG. 6 are photomicrographs showing cells 2 weeks after the start of culture. Fig. 5 is a photomicrograph of the nonwoven fabric (Example 3) to which the hydroxyapatite is fixed, and Fig. 6 is a photomicrograph of the non-woven fabric (Comparative Example 1) with the hydroxyapatite attached. .
図 5及び図 6からわかるように、細胞培養用基材を用いた場合、細胞培養用基材の 多くの繊維に細胞が付着していた（図 5)のに対し、ハイドロキシアパタイトを付着させ ていない不織布を用いた場合、繊維の一部にしか細胞が付着していなかった（図 6) As can be seen from FIG. 5 and FIG. 6, when the cell culture substrate is used, the cell culture substrate Cells were attached to many fibers (Fig. 5), whereas when non-woven fabric without hydroxyapatite was used, cells were attached only to a part of the fibers (Fig. 6).
[0040] 培養開始から 1ヶ月後に細胞のコラーゲン産生量 (培養上清中のコラーゲン量）を 測定した結果、実施例 3の場合、コラーゲン産生量は 5.6 x gZml (細胞培養用基材 以外の部分に付着した細胞が産生したコラーゲン量を除いた値）であったのに対し、 比較例 1の場合、コラーゲン産生量は、 1.3 x g/ml (不織布以外の部分に付着した 細胞が産生したコラーゲン量を除レ、た値）であった。 [0040] As a result of measuring the amount of collagen produced by the cells (the amount of collagen in the culture supernatant) one month after the start of culture, in Example 3, the amount of collagen produced was 5.6 x gZml (part other than the cell culture substrate) In the case of Comparative Example 1, the amount of collagen produced was 1.3 xg / ml (the amount of collagen produced by cells attached to parts other than the non-woven fabric). Was the value).
[0041] 実施例 3と比較例 1の比較から、不織布にハイドロキシアパタイトを固着させることに より、細胞の付着及び増殖が促進されること及びコラーゲンの生産量が増大すること が分かった。  [0041] From the comparison between Example 3 and Comparative Example 1, it was found that by adhering hydroxyapatite to the nonwoven fabric, cell attachment and proliferation were promoted and collagen production was increased.
[0042] 実施例 4  [0042] Example 4
不織布の繊維密度を検討した。不織布の繊維の密度は、不織布の単位面積あたり の重量で調節できる。そこで、密度（単位面積あたりの重量）の異なる 3種類の不織布 を準備した。各不織布の単位面積（10 cm X IO cm)あたりの重量（「不織布のみ」のグ ラム数）は表 2に示す通りであった。これらの不織布を用いた以外実施例 1と同様にし て細胞培養用基材 (サンプル：!〜 3)を得、各サンプルにヒト正常線維芽細胞を 4 X 10 5個まき込んだ以外実施例 1と同様にして培養を行った。  The fiber density of the nonwoven fabric was examined. The density of the nonwoven fabric fiber can be adjusted by the weight per unit area of the nonwoven fabric. Therefore, we prepared three types of nonwoven fabrics with different densities (weight per unit area). Table 2 shows the weight per unit area (10 cm X IO cm) of each nonwoven fabric (number of grams of “nonwoven fabric only”). A cell culture substrate (sample:! To 3) was obtained in the same manner as in Example 1 except that these non-woven fabrics were used, and Example 1 except that 4 x 10 5 human normal fibroblasts were infused into each sample. Culture was performed in the same manner as described above.
対照として、サンプル 1の不織布にハイドロキシアパタイトを付着させていないものを 用いて、同様に細胞培養した。  As a control, cell culture was performed in the same manner using the non-woven fabric of Sample 1 without hydroxyapatite attached.
[0043] 表中、「不織布 + HAp」の欄は、細胞培養用基材の単位面積あたり（10 cm X IO cm )の重量を示す。 In the table, the column “nonwoven fabric + HAp” indicates the weight per unit area (10 cm × IO cm 2) of the cell culture substrate.
表中、「HAp」の欄は、その不織布に付着したハイドロキシアパタイトの重量を示す。 ノ、イドロキシアパタイトの重量は、細胞培養用基材を燃焼させ、残渣の重量を測定す ることにより求めた。  In the table, the column “HAp” indicates the weight of hydroxyapatite adhering to the nonwoven fabric. The weight of the iodoxiapatite was determined by burning the cell culture substrate and measuring the weight of the residue.
表中、「不織布のみ」の欄は、不織布の単位面積あたりの重量を示す。不織布の単 位面積あたりの重量は、「不織布 + HAp」の欄の値 (A)力 「HAp」の欄の値（B)を減 じることにより算出した。 [表 2] In the table, the column of “nonwoven fabric only” indicates the weight per unit area of the nonwoven fabric. The weight per unit area of the non-woven fabric was calculated by subtracting the value (A) force in the “non-woven fabric + HAp” column (A) and the value (B) in the “HAp” column. [Table 2]
Figure imgf000013_0001
培養開始から 1ヶ月後に、培養上清中のコラーゲン量を測定した。結果を表 3に示 す。なお表 3中のコラーゲン量は、実施例 3及び比較例 1と同様、細胞培養用基材又 はハイドロキシアパタイトを付着させていない不織布以外の部分に付着した細胞が産 生したコラーゲン量を除レ、た値である。
Figure imgf000013_0001
One month after the start of the culture, the amount of collagen in the culture supernatant was measured. The results are shown in Table 3. The amount of collagen in Table 3 is the same as in Example 3 and Comparative Example 1, except for the amount of collagen produced by cells adhering to portions other than the non-woven fabric to which the cell culture substrate or hydroxyapatite is not attached. It is a value.
[表 3]  [Table 3]
Figure imgf000013_0002
Figure imgf000013_0002
[0045] 表 3から、不織布の単位面積（10 cm X IO cm)あたりの重量が少なくとも 0.12 (0.117 188)〜0.2 (0.198804) g/100 cm2の範囲内である場合、細胞培養を良好に行うことが できることが分かった。 [0045] From Table 3, when the weight per unit area (10 cm X IO cm) of the nonwoven fabric is at least within the range of 0.12 (0.117 188) to 0.2 (0.198804) g / 100 cm 2 It turns out that it can be done.
また不織布の繊維の密度が大きい方が、培養上清中のコラーゲン量も多かった。こ れは、密度が大きい不織布の方が、付着できる細胞数が多いためであると推測する。  In addition, the higher the density of non-woven fabric fibers, the greater the amount of collagen in the culture supernatant. This is presumed to be due to the higher number of cells that can be attached to the non-woven fabric with higher density.
[0046] 実施例 5  [0046] Example 5
細胞培養用基材を 1枚又は複数枚用いて、細胞培養を行った。 75 cm2のフラスコに 培地を入れた後、細胞培養用基材を 1枚又は複数枚重ねて入れ、ヒト正常線維芽細 胞は 5 X 105個まき込み、 4〜5日間隔で培地交換を行った以外実施例 1と同様にし て培養を行った。 Cell culture was performed using one or more cell culture substrates. Place the culture medium in a 75 cm 2 flask, add one or more cell culture substrates, and infuse 5 × 105 human normal fibroblasts, and change the medium every 4 to 5 days. The culture was performed in the same manner as in Example 1 except that the above was performed.
[0047] 培養開始から 3力月後に培養上清中のコラーゲン量を測定した。結果を表 4に示す [表 4] 用いた細胞培養用基材の枚数 コラーゲン量 [0047] Collagen content in the culture supernatant was measured 3 months after the start of the culture. The results are shown in Table 4 [Table 4] Number of cell culture substrates used Collagen content
1枚 21 β g/mL  1 sheet 21 β g / mL
2枚 27/i g/mL  2 sheets 27 / ig / mL
3枚 34 μ / mL  3 plates 34 μ / mL
[0048] 培養に用いる細胞培養用基材の枚数を増やすと、少なくとも 3枚まではコラーゲン 産生量が順次増加した。このことから、細胞培養用基材を 2枚又は 3枚重ねた培養環 境は、培養細胞の付着及び Z又は増殖に好適であることが分かった。 [0048] When the number of cell culture substrates used for culturing was increased, the amount of collagen production gradually increased up to at least three. From this, it was found that a culture environment in which two or three cell culture substrates were stacked was suitable for adhesion and Z or proliferation of cultured cells.
[0049] 実施例 6  [0049] Example 6
細胞培養用基材をシャーレに固定した場合と、固定しない場合とを比較した。 本実験では、不織布の固定手段として、メビオールジェル (登録商標、メビオール 株式会社製、以下同じ)を用いた。メビオールジェルは、低温条件では流動性のゾノレ 状であり、転移温度以上の条件 (例えば、培養条件）ではゲル状になるため、低温条 件で混和した後、培養条件に移すことにより、細胞培養用基材を固定できる。  The case where the cell culture substrate was fixed to the petri dish was compared with the case where the substrate was not fixed. In this experiment, Meviol Gel (registered trademark, manufactured by Meviol Co., Ltd., hereinafter the same) was used as a means for fixing the nonwoven fabric. Meviol gel is fluid Zonole-like under low-temperature conditions, and gel-like under conditions above the transition temperature (for example, culture conditions). After mixing under low-temperature conditions, cells are transferred to culture conditions. A culture substrate can be fixed.
[0050] 不織布のサイズを 3cm X 7cmとした以外実施例 1と同様にして、細胞培養用基材を 作製した。この細胞培養用基材を直径の 10 cmシャーレに入れ、培地、メビオールジ エルを入れて不織布を固定し、ヒト正常線維芽細胞は 2 X 105個まき込み、 4〜5日間 隔で培地交換した以外実施例 1等と同様にして培養を行った。 [0050] A cell culture substrate was prepared in the same manner as in Example 1 except that the size of the nonwoven fabric was 3 cm X 7 cm. Put this cell culture substrate in a 10 cm diameter petri dish, put medium and meviol gel, fix the nonwoven, and infuse 2 X 105 human normal fibroblasts, and change the medium every 4-5 days Incubation was carried out in the same manner as in Example 1.
培養開始から 2ヶ月後に、培養上清中のコラーゲン量を測定した。  Two months after the start of culture, the amount of collagen in the culture supernatant was measured.
なお、メビオールジェルを入れない以外実施例 6と同様にして培養を行い、対照と した。  The culture was performed in the same manner as in Example 6 except that no mebiol gel was added, and was used as a control.
[0051] 結果を表 5に示す。  [0051] The results are shown in Table 5.
[表 5]  [Table 5]
Figure imgf000014_0001
表 5に示す通り、細胞培養用基材をシャーレに固定した方がコラーゲン量は多かつ た。この理由としては (a)細胞培養用基材をシャーレに固定しないと、細胞培養用基 材が浮遊及び/又は移動するため、シャーレ底面に付着した細胞に物理的なダメー ジを与え、細胞を死滅させること、及び (b)その死滅した細胞の影響により、生きた細 胞のコラーゲン産生量も低下することが考えられる。
Figure imgf000014_0001
As shown in Table 5, the amount of collagen was larger when the cell culture substrate was fixed to a petri dish. The reasons for this are as follows: (a) If the cell culture substrate is not fixed to the petri dish, the cell culture substrate will float and / or move. It is conceivable that the amount of collagen produced by living cells is also reduced due to the effects of the dead cells.
[0053] 実施例 7  [0053] Example 7
マイクロキャリアに細胞を付着させた後で細胞をまきこむ方法を検討した。直径 10 c mのシャーレに 2 X 106個のヒト繊維芽細胞を入れ、翌日、これにマイクロキャリア「CE LLYARD beads (ペンタックス株式会社製）」を 0.5〜2.0 g添加し、：!〜 3日間培養した 。この間に、細胞の半数以上がマイクロキャリアに付着した。細胞培養用基材に細胞 を巻き込む代わりに、細胞を付着させたマイクロキャリアを細胞培養用基材の上に吐 出した以外実施例 1等と同様にして培養を行った。  A method of encapsulating cells after attaching them to a microcarrier was investigated. Place 2 X 106 human fibroblasts in a petri dish with a diameter of 10 cm, and add 0.5-2.0 g of microcarrier "CE LLYARD beads (Pentax Co., Ltd.)" to the next day. . During this time, more than half of the cells adhered to the microcarriers. Culturing was carried out in the same manner as in Example 1 except that the microcarriers to which the cells were attached were discharged onto the cell culture substrate instead of entraining the cells in the cell culture substrate.
[0054] その結果、実施例 1では、コラーゲン産生量が 20 μ g/ml以上になるまでに約 2ヶ月 間かかったのに対し、本実験では、約 3週間で、コラーゲン産生量が同量以上になつ た。この結果は、通常のようにトリプシン処理して細胞を浮遊させた後まき込むより、 細胞をマイクロキャリアに付着させた後で細胞培養用基材にまき込む方が、細胞の定 着及び/又は増殖を速くできることを示す。  [0054] As a result, in Example 1, it took about 2 months for the collagen production to reach 20 μg / ml or more, whereas in this experiment, the same amount of collagen was produced in about 3 weeks. That's it. This result shows that cell attachment and / or cell culture is more likely to occur when cells are attached to a microcarrier and then inoculated into a cell culture substrate, rather than after trypsinization and floating the cells as usual. Shows that growth can be accelerated.
[0055] 実施例 8  [0055] Example 8
細胞培養用基材を用いた細胞培養に適用可能な細胞の種類を検討した。不織布 のサイズを 3cm X 7cmとした以外実施例 1と同様にして、細胞培養用基材を作製した 。底面積 25 cm2のフラスコに細胞培養用基材及び培地を入れ、巻き込む細胞を表 6 に示すとおりとした以外実施例 1と同様にして培養を行った。培養は 3ヶ月以上継続 した。 Cell types applicable to cell culture using a cell culture substrate were examined. A cell culture substrate was prepared in the same manner as in Example 1 except that the size of the nonwoven fabric was 3 cm × 7 cm. A cell culture substrate and medium were placed in a flask having a bottom area of 25 cm 2 and cultured in the same manner as in Example 1 except that the cells involved were as shown in Table 6. Incubation continued for more than 3 months.
[0056] その結果、表 6に示す通り、実験を行った全ての細胞において培養を行うことができ た。また細胞の形態や増殖速度を見る限り、培地変更等の環境変化に伴う影響もほ とんど観察されなかった。  [0056] As a result, as shown in Table 6, it was possible to culture in all the cells in which the experiment was performed. As far as the cell morphology and growth rate were concerned, almost no effects due to environmental changes such as medium changes were observed.
[表 6] 細胞名又は組織名 細胞の性状 細胞数 ヒ ト正常繊維芽細胞 細長い 多数 ヒ ト正常表皮細胞 敷石状 多数 マウス正常骨芽細胞 細長い 多数 [Table 6] Cell name or tissue name Cell properties Cell number Human normal fibroblasts Elongated many Human normal epidermal cells Paving stones Many Mouse normal osteoblasts Elongated many
O P - 9 多数  O P-9 Numerous
N I H 3 T 3 敷石状 多数  N I H 3 T 3
CMT - 9 3 粒状で膜状に拡がらない 少数 マウス大脳 塊状 少数 マウス甲状腺 粒状の膜 多数 マウス心臓 鱗状 少数 マウス肺 膜状 多数 マウス肝臓 粒状の膜 多数 マウス脾臃 少数 マウス筋肉 膜状 多数 マウス腎臓 塊で膜状 多数 マウス骨髄 粒状の膜 多数  CMT-9 3 Granular and does not spread into membranes Small number Mouse cerebrum Mass Small number Mouse thyroid Granular membrane Numerous Mouse heart Scaling Small number Mouse lung Membrane numerous Mouse liver Granular membrane Many Mouse splenic sputum Small number Mouse muscle Membrane Mass Mouse kidney Mass Membranes in large numbers Mouse bone marrow Granular membranes in large numbers
[0057] 表 6に示す培養結果から、本発明の培養方法は、ヒト正常線維芽細胞ば力りでなく 様々な種類の細胞に適用可能であることが分かった。 [0057] From the culture results shown in Table 6, it was found that the culture method of the present invention can be applied to various types of cells, not human normal fibroblasts.
産業上の利用可能性  Industrial applicability
[0058] 本発明の細胞培養用基材は、ほとんど全ての付着細胞の培養に適用できる。また 本発明の細胞培養用基材を用いると、長期間連続培養が可能であるという利点があ る。 [0058] The cell culture substrate of the present invention can be applied to culture of almost all adherent cells. In addition, when the cell culture substrate of the present invention is used, there is an advantage that long-term continuous culture is possible.

Claims

請求の範囲 The scope of the claims
[1] シート状の細胞培養用基材であって、リン酸カルシウム及び繊維からなることを特徴 とする細胞培養用基材。  [1] A substrate for cell culture, which is a sheet-shaped substrate for cell culture, comprising calcium phosphate and fibers.
[2] 請求項 1に記載の細胞培養用基材において、前記リン酸カルシウムによって覆わ れていることを特徴とする細胞培養用基材。 [2] The cell culture substrate according to claim 1, wherein the cell culture substrate is covered with the calcium phosphate.
[3] 請求項 1又は 2に記載の細胞培養用基材において、前記繊維が不織布を形成して レ、ることを特徴とする細胞培養基材。 [3] The cell culture substrate according to claim 1 or 2, wherein the fibers form a nonwoven fabric.
[4] 請求項 1〜3のいずれかに記載の細胞培養用基材において、前記リン酸カルシゥ ムがハイドロキシアパタイトであることを特徴とする細胞培養用基材。 [4] The cell culture substrate according to any one of claims 1 to 3, wherein the calcium phosphate is hydroxyapatite.
[5] 請求項 1〜4のいずれかに記載の細胞培養用基材において、三次元培養に用いら れることを特徴とする細胞培養用基材。  5. The cell culture substrate according to any one of claims 1 to 4, wherein the cell culture substrate is used for three-dimensional culture.
[6] 請求項 2〜5のいずれかに記載の細胞培養用基材において、前記不織布の単位 面積あたりの重量が、 0.1-0.3 g/100 cm2であることを特徴とする細胞培養用基材。 [6] In cell culture substrate according to any one of claims 2 to 5, weight per unit area of the nonwoven fabric, 0.1-0.3 g / 100 cm 2 cell culture group, which is a Wood.
[7] 請求項:!〜 6のいずれかに記載の細胞培養用基材を具備する培養用器具。 [7] Claims: A culture instrument comprising the cell culture substrate according to any one of! To 6.
[8] 請求項 1〜6のいずれかに記載の細胞培養用基材又は請求項 7に記載の培養用 器具を用いる細胞培養方法。 [8] A cell culture method using the cell culture substrate according to any one of claims 1 to 6 or the culture instrument according to claim 7.
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