WO2004005454A1 - Cell culture apparatus using hydroxyapatite and cell module - Google Patents

Cell culture apparatus using hydroxyapatite and cell module Download PDF

Info

Publication number
WO2004005454A1
WO2004005454A1 PCT/JP2003/008471 JP0308471W WO2004005454A1 WO 2004005454 A1 WO2004005454 A1 WO 2004005454A1 JP 0308471 W JP0308471 W JP 0308471W WO 2004005454 A1 WO2004005454 A1 WO 2004005454A1
Authority
WO
WIPO (PCT)
Prior art keywords
carrier
cell culture
peripheral wall
cell
culture
Prior art date
Application number
PCT/JP2003/008471
Other languages
French (fr)
Japanese (ja)
Inventor
Yoichi Ishikawa
Tetsuro Ogawa
Original Assignee
Able Corporation
Pentax Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Able Corporation, Pentax Corporation filed Critical Able Corporation
Priority to AU2003246256A priority Critical patent/AU2003246256A1/en
Publication of WO2004005454A1 publication Critical patent/WO2004005454A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters

Definitions

  • the present invention relates to a cell culture device and a cell module, and more particularly, to a cell culture device effective for culturing animal cells at high density in a carrier.
  • Cell modules are used effectively in many fields, such as production of useful substances, safety evaluation of food and medicine, artificial organs and tissues, and pharmacology. Background art
  • a reactor has been put into practical use in which a culture solution flows in a reverse radial direction.
  • This reactor is an excellent reactor capable of uniformly supplying a culture solution to cells growing inside the carrier, on the surface of the carrier, or in the gap between the carriers, if the particulate carrier is uniformly and closely packed.
  • the present invention has been made in view of such problems of the related art, does not require a double-arranged cylindrical net for accommodating the carrier, does not require an operation of filling the carrier, Further, it is an object of the present invention to provide a cell culture device and a cell module on which cells are formed, which can observe a carrier during culturing (the present inventors have conducted intensive studies to solve the above problems, It has been found that if the porous carrier having a specific shape and containing a specific material is formed in advance in a block shape, it is not necessary to fill the carrier in the form of particles, and the above-mentioned problem can be solved. Thus, the present invention has been completed.
  • the cell culture apparatus of the present invention comprises a porous and rigid cylindrical cell culture carrier containing hydroxyapatite, a cylindrical envelope surrounding the carrier via a space, and the culture solution in the space. And a culture solution circulating means for circulating in a direction toward the center through the outer peripheral wall surface of the carrier.
  • the peripheral wall of the cell culture carrier has a thickness of 2 cm or less. If it exceeds 2 cm, when the cells grow at a high density, dissolved oxygen will be lost downstream of the cell culture carrier, and the cells may be damaged.
  • the outer peripheral wall and the inner or inner peripheral wall of the cell culture carrier have a larger pressure loss than the peripheral wall of the carrier. Characterized by being coated with a porous layer.
  • the cell module of the present invention is characterized in that cells are formed on the cell culture carrier as described above, and in a preferred embodiment, the cells are formed by using the cell culture apparatus as described above. It is characterized by having done.
  • the above-mentioned carrier has rigidity and integrality, the handling of the carrier after cell culture is improved.
  • a transparent cylindrical envelope surrounding the outer peripheral wall of the carrier through a space is arranged, and the carrier is cultivated from the space in a reverse radial manner (radially toward the center).
  • the outer peripheral wall surface of the carrier can be visually recognized, and the culture state can be observed.
  • the cell culture apparatus of the present invention comprises a porous, rigid, cylindrical cell culture carrier containing hydroxyapatite, a cylindrical envelope surrounding the carrier via a space, and Culture medium flowing means for flowing from the space toward the center via the outer peripheral wall surface of the carrier.
  • the carrier has such a rigidity that it is not deformed when it is attached to or detached from the cell culture device.
  • the porosity is also affected by the cells to be cultured and the type of culture solution (medium), but usually, a porosity of 30 to 85 Vo 1% It is enough.
  • the porosity is less than 30 Vo 1%, the volumetric efficiency is not + min. If the porosity exceeds 85 Vo 1%, the pressure loss of the porous carrier becomes small, and the culture solution does not flow through the entire carrier. There is a tendency that only the part with a small pressure loss flows.
  • the porosity can be measured by applying a normal bulk density measurement method.
  • the thickness of the peripheral wall of the carrier is preferably 2 cm or less.
  • the thickness of the peripheral wall is also affected by the cells to be cultured and the type of culture solution, but if it exceeds 2 cm, dissolved oxygen will be lost downstream, that is, at the center, which may damage animal cells, which is not preferable.
  • the lower limit of the thickness of the peripheral wall of the carrier is preferably 2 mm from the viewpoint of mechanical strength.
  • the above-mentioned cell culture carrier contains hydroxyapatite.
  • Hydroxyapatite is widely used for artificial bones and artificial tooth roots, and has a property that cells easily adhere to it. It can be preferably used.
  • the above-mentioned carrier for cell culture can be produced by integrally molding using hydroxyapatite as a molding raw material, using aggregates and other additives, and firing it.
  • appropriate rigidity is imparted, so that it can be easily attached to an envelope or a housing and does not deform, so that stable culture can be performed and good handling properties can be exhibited.
  • the cell culture carrier has rigidity as described above, the culture solution can be effectively flowed into the carrier by mechanically attaching packing to the upper and lower surfaces of the carrier.
  • the cell culture carrier has a communication hole of 300 ⁇ m or less, Since animal cells are unlikely to form on the surface and do not become clogged, a flow path for the culture solution can be secured even if the animal cells proliferate.
  • the flow path becomes narrower as the position approaches the center of the carrier, and the pressure loss increases. Therefore, the effect that the culture solution flows through the entire carrier is obtained.
  • FIG. 1 is a cross-sectional view showing one example of the cell culture reactor of the present invention
  • FIG. 2 is a configuration diagram showing one example of the cell culture system of the present invention.
  • FIG. 1 is a cross-sectional view showing one example of the cell culture reactor of the present invention.
  • the bottom plate 1 is formed integrally with the central column 2.
  • an elastic sheet 3 disposed on the upper surface of the bottom plate 1 is in contact with the elastic sheet 3.
  • An integrally formed cylindrical porous carrier block 4 and a glass cylinder 5 are disposed, and the elastic sheet 6 and the top plate 7 are in contact with the upper end thereof. Are located.
  • the bottom plate 1, the top plate 7, the elastic sheets 3, 6 and the glass cylinder 5 form a substantially cylindrical envelope.
  • the female screw 9 of the culture solution outlet nozzle 8 is screwed into the male screw 10 formed at the upper end of the column 2 so that the bottom is formed.
  • the plate 1, the elastic sheets 3 and 6, the porous carrier 4, the glass cylinder 5, and the culture solution outlet nozzle 8 are fixed to the body.
  • a culture solution supply nozzle 11 is fixed to the top plate 7 by being sealed with an O-ring 12.
  • the O-ring 13 prevents the culture solution from leaking from the threaded portion between the male screw 10 and the culture solution outlet nozzle 8.
  • the glass cylinder 5 and the outer peripheral wall surface of the porous carrier 4 are separated from each other, and a space 14 is formed and communicates with the culture solution supply nozzle 11.
  • the culture solution When the culture solution is supplied from the culture solution supply nozzle 11 by a pump, the culture solution is filled in the space 14, flows from the outer periphery of the cylindrical carrier 4 in a reverse radial direction in the direction of the arrow 23, and flows into the center hollow portion 21. To reach. A part of the column 2 is thin, from which the culture solution is introduced into the hollow portion 21 in the center of the column 2 through a plurality of lateral holes 22 and discharged from the culture solution outlet nozzle 8.
  • the porous carrier 4 is a fired body containing hydroxyapatite.
  • FIG. 2 is a configuration diagram showing an example of a culture system using the cell culture reactor of the present invention.
  • a culture medium 33 in a medium adjustment tank 32 is circulated to a reactor 31 in FIG. 1 by a pump 34.
  • the pH, dissolved oxygen, dissolved carbon dioxide, and composition of the culture solution are controlled by ordinary methods. An example of culture using this cell culture device is described below.
  • a method for seeding cells in addition to the above operation, a method in which the porous carrier used in the present invention is immersed in a cell suspension, or a method in which the porous carrier is immersed in a cell suspension in a closed vessel, By applying a negative pressure, a method can be employed in which air in the carrier is evacuated and cells are distributed in the carrier.
  • the cells When the cells are seeded on the carrier, the cells are allowed to stand and adhere, and the post-culture solution is allowed to flow, so that the cells are prevented from flowing out of the carrier.
  • the porous layer disposed on the outer peripheral wall or the inner peripheral wall may be in close contact with the porous carrier, but may not be in close contact as long as it is present in the flow path through which the culture solution flows.
  • the column 2 of the above embodiment may be formed of a dense porous material having a hollow portion. In this case, the side hole 22 is unnecessary.
  • a liquid dispersion layer may be provided in close contact with the outer peripheral wall of the porous carrier or on the outer edge.
  • the liquid dispersion layer use a porous material or a cylinder with fine holes.
  • a porous carrier having a predetermined shape and containing a specific material is formed in a block shape in advance, so that a double carrier for accommodating the carrier is provided.
  • the present invention provides a cell culture device which does not require a cylindrical mesh arranged in a cell, does not require an operation of filling the carrier, and allows the carrier to be observed during culture, a cell culture carrier, and a cell module on which cells are formed. be able to.
  • the cell culture device of the present invention allows buoyant or adherent cells to be cultured together.
  • This culture device can be used for culturing CHO, hepatic parenchymal cells, hepatic non-parenchymal cells, endothelial cells, and knees.
  • the present invention can be applied to culture of various cells such as cells, keratinocytes, fibroblasts, and co-culture thereof.
  • hepatocytes are responsible for many metabolic functions in vivo
  • the cell module constructed by culturing hepatocytes at high density according to the present invention can be used for material production, food, drug safety evaluation, and liver function evaluation.
  • the present invention is also effective as a device for introducing a virus such as hepatitis or its gene into the cells of the module to replicate the virus.

Abstract

A cell culture apparatus which comprises a cylindrical cell culture support containing hydroxyapatite and having porosity and rigidity, a cylindrical envelope surrounding the support via a space, and a liquid culture medium-flowing means for supplying a liquid culture medium from the space toward the center via the outer peripheral wall of the support. The thickness of the peripheral wall of the support is 2 cm or less. A cell module comprising cells planted on the cell culture support.

Description

明細書 ヒ ドロキシァパタイ トを用いた細胞培養装置及び細胞モジュール 技術分野  TECHNICAL FIELD Cell culture apparatus and cell module using hydroxyapatite
本発明は、 細胞培養装置及び細胞モジュールに係り、 更に詳細には、 担体内に動物細胞を高密度に培養するのに有効な細胞培養装置に関する また、 培養して細胞、 特に動物細胞を担持した細胞モジュールは、 有用 物質の生産、 食品 · 薬品の安全性評価、 人工臓器や人工組織、 薬理等の 多くの方面に有効に利用される。 背景技術  The present invention relates to a cell culture device and a cell module, and more particularly, to a cell culture device effective for culturing animal cells at high density in a carrier. Cell modules are used effectively in many fields, such as production of useful substances, safety evaluation of food and medicine, artificial organs and tissues, and pharmacology. Background art
従来、 細胞培養リアクターと して、 円筒状をなす大小の網を二重に配 置し、 これらに囲まれた空間に粒子状の多孔質担体を充填し、 大きな網 の外周側から中心に向かって逆放射状に培養液を流すリアクターが実用 化されている。 このリアクターは、 粒子状担体を均一且つ最密に充填す れば、 担体内部、 担体表面や担体の隙間に増殖する細胞に培養液を均一 に供給し得る優れたリアクターである。  Conventionally, as a cell culture reactor, cylindrical large and small meshes are arranged in double, and the space surrounded by these is filled with a particulate porous carrier, and the large mesh is moved from the outer periphery to the center. A reactor has been put into practical use in which a culture solution flows in a reverse radial direction. This reactor is an excellent reactor capable of uniformly supplying a culture solution to cells growing inside the carrier, on the surface of the carrier, or in the gap between the carriers, if the particulate carrier is uniformly and closely packed.
しかしながら、 かかるリアクターは、 担体を充填する空間を形成する のに二重に配置した円筒状の網を必要と し、 構造が複雑であること、 担 体の充填に粗密があると粗の部分に培養液が流れてしまうので担体全体 として均一に充填することが必要であるが、 担体が粒子状であるため、 その充填操作が難しいこと、 更には、 円筒状の網内部に担体を配置しな ければならないので、 培養中に担体を観察することができないこと等の 問題点があった。  However, such a reactor requires a double-arranged cylindrical net to form a space for filling the carrier, and the structure is complicated. Since the culture solution flows, it is necessary to uniformly fill the carrier as a whole.However, the filling operation is difficult because the carrier is particulate, and furthermore, the carrier must not be placed inside the cylindrical mesh. Therefore, there was a problem that the carrier could not be observed during the culture.
また、 従来は上述したリアクターを用いて細胞を培養していたため、 担体と しても粒子状の担体が市販されているだけであり、 他形状の担体 が流通しておらず、 まして細胞を着生させた担体は市販されていなかつ た。 Conventionally, cells were cultured using the above-mentioned reactor, Only carriers in the form of particles are commercially available as carriers, and carriers of other shapes are not in circulation. Further, carriers on which cells have formed are not commercially available.
なお、 上述のリアクターを用いた細胞培養では、 細胞が着生した担体 を二重の円筒に囲まれた部分から取り出すのにも苦労していた。 発明の開示  In the cell culture using the above-described reactor, it was difficult to remove the carrier on which the cells had formed from the portion surrounded by the double cylinder. Disclosure of the invention
本発明は、 このような従来技術の有する課題に鑑みてなされたもので あり、 担体を収納するための二重に配置した円筒状の網が不要で、 担体 を充填する操作が不要であり、 且つ培養中に担体を観察し得る細胞培養 装置及び細胞を着生させた細胞モジュールを提供することを目的とする ( 本発明者らは上記課題を解決するため鋭意検討を重ねた結果、 所定の 形状を有し特定材料を含有する多孔質担体を予めプロック状に形成して おけば粒子状の担体を充填する必要がなく、 上記課題を解決できること を見出し、 本発明を完成するに至った。 The present invention has been made in view of such problems of the related art, does not require a double-arranged cylindrical net for accommodating the carrier, does not require an operation of filling the carrier, Further, it is an object of the present invention to provide a cell culture device and a cell module on which cells are formed, which can observe a carrier during culturing (the present inventors have conducted intensive studies to solve the above problems, It has been found that if the porous carrier having a specific shape and containing a specific material is formed in advance in a block shape, it is not necessary to fill the carrier in the form of particles, and the above-mentioned problem can be solved. Thus, the present invention has been completed.
即ち、 本発明の細胞培養装置は、 ヒ ドロキシァパタイ トを含有する多 孔質で剛性を有する円筒状の細胞培養担体と、 この担体を空間を介して 包囲する円筒形エンベロープと、 培養液を上記空間から上記担体の外周 壁面を介して中心部方向に流通させる培養液流通手段と、 を備えること を特徴とする。  That is, the cell culture apparatus of the present invention comprises a porous and rigid cylindrical cell culture carrier containing hydroxyapatite, a cylindrical envelope surrounding the carrier via a space, and the culture solution in the space. And a culture solution circulating means for circulating in a direction toward the center through the outer peripheral wall surface of the carrier.
また、 本発明の細胞培養装置の好適形態は、 上記細胞培養担体の周壁 の厚さが 2 c m以下であることを特徴とする。 2 c mを超えると、 細胞 が高密度に増殖したとき、 細胞培養担体の下流で溶存酸素がなくなり、 細胞に損傷を与えるおそれがある。  In a preferred embodiment of the cell culture device of the present invention, the peripheral wall of the cell culture carrier has a thickness of 2 cm or less. If it exceeds 2 cm, when the cells grow at a high density, dissolved oxygen will be lost downstream of the cell culture carrier, and the cells may be damaged.
更に、 本発明の細胞培養装置の他の好適形態は、 上記細胞培養担体の 外周壁面及ぴノ又は内周壁面に、 この担体の周壁より も圧力損失の大き な多孔質層を被覆して成ることを特徴とする。 Further, another preferred embodiment of the cell culture apparatus of the present invention is that the outer peripheral wall and the inner or inner peripheral wall of the cell culture carrier have a larger pressure loss than the peripheral wall of the carrier. Characterized by being coated with a porous layer.
また、 本発明の細胞モジュールは、 上述の如き細胞培養担体に、 細胞 を着生させて成ることを特徴と し、 その好適形態は、 上記細胞の着生を、 上述の如き細胞培養装置を用いて行ったことを特徴とする。  Further, the cell module of the present invention is characterized in that cells are formed on the cell culture carrier as described above, and in a preferred embodiment, the cells are formed by using the cell culture apparatus as described above. It is characterized by having done.
本発明の細胞培養装置では、 一体的に形成された円筒状の多孔質担体 を用いるので、 従来のように粒子状担体を収納するための二重に配置し た円筒状の網が不必要である。  In the cell culture apparatus of the present invention, since a cylindrical porous carrier formed integrally is used, a double cylindrical cylinder for accommodating the particulate carrier as in the related art is unnecessary. is there.
また、 均質な上記多孔質担体さえ入手できれば、 粒子状担体を充填す る煩雑な操作が省略されるばかりか、 粒子状担体を充填することにより 発生するおそれのある担体充填の粗密を回避することができ、 均一で再 現性の高い細胞培養が可能になる。  In addition, if a homogeneous porous carrier can be obtained, the complicated operation of filling the particulate carrier is not only omitted, but also the packing of the carrier which may be generated by filling the particulate carrier is avoided. This allows for uniform and highly reproducible cell culture.
更には、 上記担体が剛性及び一体性を有することから、 細胞培養後の 担体の取り出しゃ取扱性も改善される。  Furthermore, since the above-mentioned carrier has rigidity and integrality, the handling of the carrier after cell culture is improved.
また、 円筒状の網が不要なので、 上記担体の外周壁を、 空間を介して 取り囲む透明な円筒形エンベロープを配置し、 かかる空間から該担体に 逆放射状 （中心部へ向かって半径方向） に培養液を供給すれば、 該担体 の外周壁面を視認することができ、 培養状態を観察することが可能にな る。  In addition, since a cylindrical mesh is not necessary, a transparent cylindrical envelope surrounding the outer peripheral wall of the carrier through a space is arranged, and the carrier is cultivated from the space in a reverse radial manner (radially toward the center). When the liquid is supplied, the outer peripheral wall surface of the carrier can be visually recognized, and the culture state can be observed.
以下、 本発明について詳細に説明する。 なお、 本明細書において、 「％」 は特 f己しない限り質量百分率を表す。  Hereinafter, the present invention will be described in detail. In addition, in this specification, "%" represents a mass percentage unless otherwise specified.
上述の如く、 本発明の細胞培養装置は、 多孔質で剛性があり ヒ ドロキ シァパタイ トを含む円筒状の細胞培養担体と、 この担体を空間を介して 包囲する円筒形エンベロープと、 培養液を上記空間から上記担体の外周 壁面を介して中心部方向に流通させる培養液流通手段と、 を備える。  As described above, the cell culture apparatus of the present invention comprises a porous, rigid, cylindrical cell culture carrier containing hydroxyapatite, a cylindrical envelope surrounding the carrier via a space, and Culture medium flowing means for flowing from the space toward the center via the outer peripheral wall surface of the carrier.
ここで、 上述の細胞培養担体の剛性については、 細胞培養装置への装 着 ■脱着等において変形しない程度の剛性を有すれば十分である。 また、 多孔質性と しては、 培養対象となる細胞やその培養液 （培地） の種類などにも影響を受けるが、 通常は 3 0〜 8 5 V o 1 %の気孔率を 有すれば十分である。 Here, as for the rigidity of the above-mentioned cell culture carrier, it is sufficient that the carrier has such a rigidity that it is not deformed when it is attached to or detached from the cell culture device. The porosity is also affected by the cells to be cultured and the type of culture solution (medium), but usually, a porosity of 30 to 85 Vo 1% It is enough.
気孔率が 3 0 V o 1 %未満では、 体積効率が +分ではなく、 8 5 V o 1 %を超えると、 多孔質担体の圧力損失が小さくなり、 培養液が担体全 体に流れず、 圧力損失の小さい部分だけを流れてしまう傾向がある。 な お、 かかる気孔率は、 通常のかさ密度測定法を適用して測定することが できる。  If the porosity is less than 30 Vo 1%, the volumetric efficiency is not + min.If the porosity exceeds 85 Vo 1%, the pressure loss of the porous carrier becomes small, and the culture solution does not flow through the entire carrier. There is a tendency that only the part with a small pressure loss flows. The porosity can be measured by applying a normal bulk density measurement method.
更に、 担体の周壁の厚さは 2 c m以下であることが好ましい。 周壁の 厚さも、 培養対象たる細胞や培養液の種類などにも影響を受けるが、 2 c mを超えると、 下流すなわち中心部分で溶存酸素がなくなり、 動物細 胞に損傷を与えることがあり好ましくない。 なお、 担体周壁の厚さの下 限値は、 機械的強度の面から 2 m mであることが好ましい。  Further, the thickness of the peripheral wall of the carrier is preferably 2 cm or less. The thickness of the peripheral wall is also affected by the cells to be cultured and the type of culture solution, but if it exceeds 2 cm, dissolved oxygen will be lost downstream, that is, at the center, which may damage animal cells, which is not preferable. . The lower limit of the thickness of the peripheral wall of the carrier is preferably 2 mm from the viewpoint of mechanical strength.
また、 上述の細胞培養担体は、 ヒ ドロキシアパタイ トを含有するが、 ヒ ドロキシァパタイ トは人工骨や人工歯根にも広く利用されており、 細 胞が接着し易い性質を有するので細胞培養担体と して好ましく使用する ことができる。  In addition, the above-mentioned cell culture carrier contains hydroxyapatite.Hydroxyapatite is widely used for artificial bones and artificial tooth roots, and has a property that cells easily adhere to it. It can be preferably used.
本発明において、 上記細胞培養担体は、 ヒ ドロキシァパタイ トを成形 原料と して、 骨材その他の添加剤を用いて一体成形し、 焼成することに より製造することができる。 このように一体的に形成しておけば適切な 剛性が付与されるため、 エンベロープやハウジングに装着し易く変形も しないので、 安定な培養が可能であり良好な取扱い性を発揮し得る。 なお、 上述のように細胞培養担体が剛性を有するので、 この担体の上 面と下面にパッキンを機械的に密着させることによって、 担体内部に培 養液を有効に流すことができる。  In the present invention, the above-mentioned carrier for cell culture can be produced by integrally molding using hydroxyapatite as a molding raw material, using aggregates and other additives, and firing it. When formed integrally in this manner, appropriate rigidity is imparted, so that it can be easily attached to an envelope or a housing and does not deform, so that stable culture can be performed and good handling properties can be exhibited. Since the cell culture carrier has rigidity as described above, the culture solution can be effectively flowed into the carrier by mechanically attaching packing to the upper and lower surfaces of the carrier.
また、 細胞培養担体が 3 0 0 μ m以下の連通孔を有すると、 その内部 には動物細胞が着生し難く、 目詰まりすることがないので、 動物細胞が 増殖しても培養液の流路を確保することができる。 If the cell culture carrier has a communication hole of 300 μm or less, Since animal cells are unlikely to form on the surface and do not become clogged, a flow path for the culture solution can be secured even if the animal cells proliferate.
また、 本発明においては、 細胞培養担体に培養液を逆放射状に流すた め、 担体の中心に近付くほど流路が狭くなつて圧力損失が大きく るので、 培養液が担体全体を流れる効果が得られる。  Further, in the present invention, since the culture solution flows in the reverse direction in the cell culture carrier, the flow path becomes narrower as the position approaches the center of the carrier, and the pressure loss increases. Therefore, the effect that the culture solution flows through the entire carrier is obtained. Can be
更に、 かかる細胞培養担体の外周壁面及び z又は内周壁面に、 担体材 質より も圧力損失の大きな多孔質層を被覆することが可能であり、 これ により、 培養液をより担体全体に流すことができ、 担体全体で細胞を培 養することが可能になる。 図面の簡単な説明  Furthermore, it is possible to coat the outer peripheral wall and z or inner peripheral wall of such a cell culture carrier with a porous layer having a greater pressure loss than the carrier material, thereby allowing the culture solution to flow over the entire carrier. This allows cells to be cultured on the entire carrier. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 本発明の細胞培養リアクターの一例を示す断面図、 図 2は、 本発明の細胞培養システムの一例を示す構成図である。 発明を実施するための最良の形態  FIG. 1 is a cross-sectional view showing one example of the cell culture reactor of the present invention, and FIG. 2 is a configuration diagram showing one example of the cell culture system of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明を若干の実施例により更に詳細に説明するが、 本発明は これら実施例に限定されるものではない。  Hereinafter, the present invention will be described in more detail with reference to some examples, but the present invention is not limited to these examples.
(実施例 1 )  (Example 1)
図 1は、 本発明の細胞培養リアクターの一例を示す断面図である。 この細胞培養リアクター 3 1において、 底板 1は中央の柱 2 と一体に 形成されている。 また、 底板 1の上面に配置した弾性シート 3に接して. 一体形成された円筒状の多孔質担体プロック 4及びガラス円筒 5が配置 され、 その上端に接して弾性シート 6、 更に天板 7が配置されている。 これら底板 1、 天板 7、 弾性シート 3 , 6及びガラス円筒 5は、 ほぼ円 筒形のエンベロープを形成している。 そして、 培養液出口ノズル 8の雌 ねじ 9を柱 2の上端に形成した雄ねじ 1 0に螺合することによって、 底 板 1、 弾性シート 3及ぴ 6、 多孔質担体 4、 ガラス円筒 5及び培養液出 口ノズル 8がー体に固定されている。 FIG. 1 is a cross-sectional view showing one example of the cell culture reactor of the present invention. In this cell culture reactor 31, the bottom plate 1 is formed integrally with the central column 2. In addition, an elastic sheet 3 disposed on the upper surface of the bottom plate 1 is in contact with the elastic sheet 3. An integrally formed cylindrical porous carrier block 4 and a glass cylinder 5 are disposed, and the elastic sheet 6 and the top plate 7 are in contact with the upper end thereof. Are located. The bottom plate 1, the top plate 7, the elastic sheets 3, 6 and the glass cylinder 5 form a substantially cylindrical envelope. Then, the female screw 9 of the culture solution outlet nozzle 8 is screwed into the male screw 10 formed at the upper end of the column 2 so that the bottom is formed. The plate 1, the elastic sheets 3 and 6, the porous carrier 4, the glass cylinder 5, and the culture solution outlet nozzle 8 are fixed to the body.
天板 7には、 培養液供給ノズル 1 1が天板 7に Oリング 1 2で密封さ れて固定されている。 Oリング 1 3は、 雄ねじ 1 0 と培養液出口ノズル 8の螺合部から培養液が漏れるのを防いでいる。 ガラス円筒 5 と多孔質 担体 4の外周壁面とは離間しており、 空間 1 4が形成されていて培養液 供給ノズル 1 1 と連通している。  A culture solution supply nozzle 11 is fixed to the top plate 7 by being sealed with an O-ring 12. The O-ring 13 prevents the culture solution from leaking from the threaded portion between the male screw 10 and the culture solution outlet nozzle 8. The glass cylinder 5 and the outer peripheral wall surface of the porous carrier 4 are separated from each other, and a space 14 is formed and communicates with the culture solution supply nozzle 11.
次に、 この細胞培養リアクターの作用につき説明する。  Next, the operation of the cell culture reactor will be described.
培養液をポンプで培養液供給ノズル 1 1から供給すると、 培養液は空 間 1 4に満たされ、 円筒状担体 4の外周から矢印 2 3 の方向に逆放射状 に流れて中心の中空部 2 1に到達する。 柱 2の一部は細くなっていて、 培養液はそこから柱 2の中心部の中空部 2 1に複数の横穴 2 2を通って 導入され、 培養液出口ノズル 8から排出される。  When the culture solution is supplied from the culture solution supply nozzle 11 by a pump, the culture solution is filled in the space 14, flows from the outer periphery of the cylindrical carrier 4 in a reverse radial direction in the direction of the arrow 23, and flows into the center hollow portion 21. To reach. A part of the column 2 is thin, from which the culture solution is introduced into the hollow portion 21 in the center of the column 2 through a plurality of lateral holes 22 and discharged from the culture solution outlet nozzle 8.
次に、 上述したリアクターの構成要素の材質などにつき説明すると、 多孔質担体 4は、 ヒ ドロキシァパタイ トを含有する焼成体である。  Next, the material of the components of the reactor described above will be described. The porous carrier 4 is a fired body containing hydroxyapatite.
(実施例 2 )  (Example 2)
図 2は、 本発明の細胞培養リァクターを用いた培養システムの一例を 示す構成図である。  FIG. 2 is a configuration diagram showing an example of a culture system using the cell culture reactor of the present invention.
この培養システムでは、 図 1 のリアクター 3 1に培地調整槽 3 2の培 地 3 3をポンプ 3 4で循環する。 培地 3 3は p H、 溶存酸素、 溶存炭酸 ガス及び培養液組成が通常の手法で制御されている。 この細胞培養装置 を用いる培養例を以下に記す。  In this culture system, a culture medium 33 in a medium adjustment tank 32 is circulated to a reactor 31 in FIG. 1 by a pump 34. In the medium 33, the pH, dissolved oxygen, dissolved carbon dioxide, and composition of the culture solution are controlled by ordinary methods. An example of culture using this cell culture device is described below.
(培養例）  (Culture example)
細胞を培地調整槽 3 2中の培地に投入しポンプ 3 4でリアクター 3 1 内に送ると、 細胞は多孔質担体 4に送られ、 播種される。 その時の流速 は多孔質担体全体に均一に播種されるよう適切に制御する必要がある。 細胞が担体内に播種された後、 必要が有れば流速を変更し培養する。 気孔率 8 0 v o 1 %、 直径 2 c m、 中空部の直径 4 mm、 担体の連通 孔の大きさと して 5 0 z m〜 3 0 0 jLi mであるヒ ドロキシァパタイ トを 担体と して C H〇一 k l (C h i n e s e H a m s t e r O v a r y ) を培養したところ、 担体体積 1 m 1 当たり 4 X 1 08個の細胞密度に 増殖した。 ここで、 細胞培養担体を取り出したところ、 動物細胞が均一 且つ高密度に育成されている細胞モジュールが形成されていた。 When cells are put into the medium in the medium adjusting tank 32 and sent into the reactor 31 by the pump 34, the cells are sent to the porous carrier 4 and seeded. At that time, it is necessary to appropriately control the flow rate so that the seed is uniformly seeded on the entire porous carrier. After the cells are seeded in the carrier, culture is performed at a different flow rate if necessary. Hydroxyapatite having a porosity of 80 vo 1%, a diameter of 2 cm, a hollow diameter of 4 mm, and a communication hole of the carrier of 50 zm to 300 jLim is used as a carrier. When kl (Chinese Hamster O vary) was cultured, it grew to a cell density of 4 × 10 8 cells / m 1 of carrier volume. Here, when the cell culture carrier was taken out, a cell module in which animal cells were grown uniformly and at high density was formed.
細胞の播種方法と しては、 上記操作のほか、 本発明で用いる多孔質担 体を細胞の懸濁液に浸漬する方法や、 多孔質担体を密閉容器中で細胞懸 濁液に浸漬し、 陰圧にすることによって担体内の空気を抜く と共に担体 内に細胞を分配する方法を採用することができる。  As a method for seeding cells, in addition to the above operation, a method in which the porous carrier used in the present invention is immersed in a cell suspension, or a method in which the porous carrier is immersed in a cell suspension in a closed vessel, By applying a negative pressure, a method can be employed in which air in the carrier is evacuated and cells are distributed in the carrier.
細胞を担体に播種した後、 静置して接着させて後培養液を流すと、 細 胞が担体から流失するのを抑制できる。  When the cells are seeded on the carrier, the cells are allowed to stand and adhere, and the post-culture solution is allowed to flow, so that the cells are prevented from flowing out of the carrier.
なお、 本発明においては、 培養液を多孔質担体内に均一に流すために 担体内に圧力損失を生ずる部分を設けることが望ましい。  In the present invention, it is desirable to provide a part that generates a pressure loss in the carrier in order to allow the culture solution to flow uniformly in the porous carrier.
即ち、 本発明で用いる円筒状の多孔質担体は、 外周壁から中心部に培 養液を流すと、 中心付近で流速が早くなり中心部での圧力損失が大きく なるので、 培養液が均一に流れやすい構造であるが、 更に均一に流すた めには、 多孔質担体の外周壁又は内周壁に上記多孔質担体を構成する材 料より も圧力損失の大きな多孔質層を配置することが好ましい。  That is, when a culture solution is flowed from the outer peripheral wall to the center of the cylindrical porous carrier used in the present invention, the flow rate increases near the center and the pressure loss at the center increases, so that the culture solution is uniform. Although the structure is easy to flow, it is preferable to arrange a porous layer having a larger pressure loss than the material constituting the porous carrier on the outer peripheral wall or inner peripheral wall of the porous carrier for more uniform flow. .
ここで、 外周壁又は内周壁に配置する多孔質層は、 多孔質担体に密着 していてもよいが、 密着していなくても培養液が流通する流路中に存在 すればよく、 例えば、 上記実施例の柱 2を中空部を有する緻密な多孔質 材で形成してもよい。 なお、 この場合、 横穴 2 2は不要である。  Here, the porous layer disposed on the outer peripheral wall or the inner peripheral wall may be in close contact with the porous carrier, but may not be in close contact as long as it is present in the flow path through which the culture solution flows. The column 2 of the above embodiment may be formed of a dense porous material having a hollow portion. In this case, the side hole 22 is unnecessary.
また、 多孔質担体の外周壁に密着して又は外縁に液分散層を設けても よい。 この液分散層と しては、 多孔質材又は微細な孔をあけた円筒を用 い得る 産業上の利用可能性 Further, a liquid dispersion layer may be provided in close contact with the outer peripheral wall of the porous carrier or on the outer edge. As the liquid dispersion layer, use a porous material or a cylinder with fine holes. Possible industrial applicability
以上説明してきたように、 本発明によれば、 所定の形状を有し特定材 料を含有する多孔質担体を予めプロック状に形成することなどと したた め、 担体を収納するための二重に配置した円筒状の網が不要で、 担体を 充填する操作が不要であり、 且つ培養中に担体を観察し得る細胞培養装 置、 細胞培養担体及び細胞を着生させた細胞モジュールを提供すること- ができる。  As described above, according to the present invention, a porous carrier having a predetermined shape and containing a specific material is formed in a block shape in advance, so that a double carrier for accommodating the carrier is provided. The present invention provides a cell culture device which does not require a cylindrical mesh arranged in a cell, does not require an operation of filling the carrier, and allows the carrier to be observed during culture, a cell culture carrier, and a cell module on which cells are formed. be able to.
例えば、 本発明の細胞培養装置によって、 浮遊性や付着性の細胞が共 に培養可能となり、 この培養装置は、 上記 C H Oの培養の他、 肝実質細 胞、 肝非実質細胞、 内皮細胞、 膝細胞、 角化細胞及び線維芽細胞やそれ らの共培養などの各種の細胞の培養に適用できる。  For example, the cell culture device of the present invention allows buoyant or adherent cells to be cultured together. This culture device can be used for culturing CHO, hepatic parenchymal cells, hepatic non-parenchymal cells, endothelial cells, and knees. The present invention can be applied to culture of various cells such as cells, keratinocytes, fibroblasts, and co-culture thereof.
特に、 肝細胞は生体内で代謝機能の多くを司っており、 本発明により 肝細胞を高密度に培養して構築した細胞モジュールは、 物質生産、 食品 ■薬品の安全性評価、 肝機能評価等の他、 モジュールの細胞に肝炎等の ゥィルス又はその遺伝子を導入してウィルスを複製させる装置と しても 有効である。  In particular, hepatocytes are responsible for many metabolic functions in vivo, and the cell module constructed by culturing hepatocytes at high density according to the present invention can be used for material production, food, drug safety evaluation, and liver function evaluation. In addition, the present invention is also effective as a device for introducing a virus such as hepatitis or its gene into the cells of the module to replicate the virus.

Claims

請求の範囲 The scope of the claims
1 . ヒ ドロキシアパタイ トを含有する多孔質で剛性を有する円筒状の細 胞培養担体と、 この担体を空間を介して包囲する円筒形エンベロープと、 培養液を上記空間から上記担体の外周壁面を介して中心部方向に流通さ せる培養液流通手段と、 を備えることを特徴とする細胞培養装置。 1. A porous and rigid cylindrical cell culture carrier containing hydroxyapatite, a cylindrical envelope surrounding the carrier through a space, and a culture solution from the space through the outer peripheral wall of the carrier. And a culture solution distribution means for causing the culture solution to flow in the direction of the center.
2 . 上記細胞培養担体の周壁の厚さが 2 c m以下であることを特徴とす る請求項 1に記載の細胞培養装置。  2. The cell culture device according to claim 1, wherein the thickness of the peripheral wall of the cell culture carrier is 2 cm or less.
3 . 上記細胞培養担体が 3 0 0 μ m以下の連通孔を有することを特徴と する請求項 1又は 2に記載の細胞培養装置。  3. The cell culture device according to claim 1, wherein the carrier for cell culture has a communication hole of 300 μm or less.
4 . 上記細胞培養担体の外周壁面及ぴ Z又は内周壁面に、 この担体の周 壁より も圧力損失の大きな多孔質層を被覆して成ることを特徴とする請 求項 1 〜 3のいずれか 1つの項に記載の細胞培養装置。  4. The method according to any one of claims 1 to 3, wherein the outer peripheral wall and the inner peripheral wall or the inner peripheral wall of the cell culture carrier are coated with a porous layer having a larger pressure loss than the peripheral wall of the carrier. The cell culture device according to one of the above items.
5 . 請求項 1 〜 4のいずれか 1つの項に記載の細胞培養担体に、 細胞を 着生させて成ることを特徴とする細胞モジュール。  5. A cell module obtained by growing cells on the cell culture carrier according to any one of claims 1 to 4.
6 . 上記細胞の着生を、 請求項 1 〜 4のいずれか 1つの項に記載の細胞 培養装置を用いて行ったことを特徴とする請求項 5に記載の細胞モジュ 6. The cell module according to claim 5, wherein the cell formation is performed using the cell culture device according to any one of claims 1 to 4.
"ノレ。 "Nore.
PCT/JP2003/008471 2002-07-05 2003-07-03 Cell culture apparatus using hydroxyapatite and cell module WO2004005454A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003246256A AU2003246256A1 (en) 2002-07-05 2003-07-03 Cell culture apparatus using hydroxyapatite and cell module

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002-196947 2002-07-05
JP2002196947 2002-07-05

Publications (1)

Publication Number Publication Date
WO2004005454A1 true WO2004005454A1 (en) 2004-01-15

Family

ID=30112382

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/008471 WO2004005454A1 (en) 2002-07-05 2003-07-03 Cell culture apparatus using hydroxyapatite and cell module

Country Status (2)

Country Link
AU (1) AU2003246256A1 (en)
WO (1) WO2004005454A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2416777A (en) * 2004-07-28 2006-02-08 Pentax Corp Cell culture apparatus
WO2019105957A1 (en) 2017-11-30 2019-06-06 Aziende Chimiche Riunite Angelini Francesco A.C.R.A.F. S.P.A. Stable liquid composition of ketoprofen, salts and enantiomers thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06277050A (en) * 1993-03-26 1994-10-04 Kanegafuchi Chem Ind Co Ltd Immobilization material for animal cell and culture method
JPH08149977A (en) * 1994-11-30 1996-06-11 Mitsubishi Materials Corp Carrier for culturing osteoblast
JPH1156343A (en) * 1997-08-21 1999-03-02 Able Kk Fixed bed reactor for culturing animal cell and sowing of cell
JP2002079288A (en) * 2000-06-27 2002-03-19 Hitachi Chem Co Ltd Open-cell cellular microorganism carrier
EP1211309A1 (en) * 1999-08-20 2002-06-05 Miyamura, Tatsuo Method and apparatus for proliferating hepatitis virus

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06277050A (en) * 1993-03-26 1994-10-04 Kanegafuchi Chem Ind Co Ltd Immobilization material for animal cell and culture method
JPH08149977A (en) * 1994-11-30 1996-06-11 Mitsubishi Materials Corp Carrier for culturing osteoblast
JPH1156343A (en) * 1997-08-21 1999-03-02 Able Kk Fixed bed reactor for culturing animal cell and sowing of cell
EP1211309A1 (en) * 1999-08-20 2002-06-05 Miyamura, Tatsuo Method and apparatus for proliferating hepatitis virus
JP2002079288A (en) * 2000-06-27 2002-03-19 Hitachi Chem Co Ltd Open-cell cellular microorganism carrier

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2416777A (en) * 2004-07-28 2006-02-08 Pentax Corp Cell culture apparatus
WO2019105957A1 (en) 2017-11-30 2019-06-06 Aziende Chimiche Riunite Angelini Francesco A.C.R.A.F. S.P.A. Stable liquid composition of ketoprofen, salts and enantiomers thereof

Also Published As

Publication number Publication date
AU2003246256A1 (en) 2004-01-23
AU2003246256A8 (en) 2004-01-23

Similar Documents

Publication Publication Date Title
US10517731B2 (en) Tissue engineering system for making personalized bone graft
EP1913126B1 (en) Co-culture bioreactor system
KR101750148B1 (en) Cell culture system and cell culture method
JP5834020B2 (en) Continuous culture equipment
WO2006095480A1 (en) Cell culture chamber
KR101566083B1 (en) circulating culture system
JP2002112763A (en) Cell culture container
JP2016202180A (en) Device and system for cell culture
JP5558560B2 (en) Bioreactor system
JP4649224B2 (en) Method and apparatus for culturing adherent cells
US20240091778A1 (en) Cell culture vessel
WO2004005454A1 (en) Cell culture apparatus using hydroxyapatite and cell module
US20100028992A1 (en) Cell culture device
US11584906B2 (en) Cell culture vessel for 3D culture and methods of culturing 3D cells
KR20210098948A (en) Modular Bioreactor
US20200040292A1 (en) Microbioreactor module
JP2023503649A (en) Radial flow fixed bed bioreactor and method of use thereof
WO2004005455A1 (en) Cell culture carrier and cell culture unit
JP7262042B2 (en) ADAPTER, CLOSED CHAMBER, CELL CULTURE DEVICE, AND ADAPTER MANUFACTURING METHOD
US20230112108A1 (en) Microcarrier based-4 dimensional cell culture apparatus and method for monitoring cell culture using the same
EP4015622A1 (en) Device for seeding cells
JPS61280270A (en) Method for cell culture and apparatus therefor
JP2024501109A (en) Cell culture medium conditioning vessels and perfusion bioreactor systems
JPH0585156B2 (en)
CN112210498A (en) Cartilage diaphragm autocrine factor retention culture dish

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP