JP4433742B2 - The method for manufacturing a culture with culture using the culture vessel, the culture vessel - Google Patents

The method for manufacturing a culture with culture using the culture vessel, the culture vessel Download PDF

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JP4433742B2
JP4433742B2 JP2003329800A JP2003329800A JP4433742B2 JP 4433742 B2 JP4433742 B2 JP 4433742B2 JP 2003329800 A JP2003329800 A JP 2003329800A JP 2003329800 A JP2003329800 A JP 2003329800A JP 4433742 B2 JP4433742 B2 JP 4433742B2
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JP2005095015A (en
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紀 幸 新谷
英 記 田所
康 二 鈴木
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株式会社ジェイ・エム・エス
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M21/00Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00Constructional details, e.g. recesses, hinges
    • C12M23/02Form or structure of the vessel
    • C12M23/04Flat or tray type, drawers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00Constructional details, e.g. recesses, hinges
    • C12M23/02Form or structure of the vessel
    • C12M23/10Petri dish
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00Constructional details, e.g. recesses, hinges
    • C12M23/30Constructional details, e.g. recesses, hinges biodegradable
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00Constructional details, e.g. recesses, hinges
    • C12M23/44Multiple separable units; Modules

Description

本発明は細胞層が多層の形で積層された積層細胞層、特に多種類の細胞層が多層の形で積層された積層細胞層を作製するために使用される培養容器、増殖用細胞が播種された前記の培養容器、増殖用細胞が播種された前記の培養容器の培養物とその培養方法、および前記培養物を使用した生体組織の再生方法に関する。 The present invention is laminated cell layers cell layers are stacked in the form of a multilayer, especially various types of culture vessels cell layer is used to produce laminated cell layers laminated in the form of a multilayer, proliferation for cell seeding the culture containers, the culture and how culture of said culture vessel for growth cells were seeded, and a method of regeneration of living tissue using the culture.

従来、組織欠損部位への移植用材料として、代表的なものに人工皮膚が挙げられる。 Conventionally, as an implant material to the tissue defect site, it includes artificial skin representative. これら細胞膜の多くはコラーゲンに代表される生体由来の吸収性材料を細胞増殖の基材として細胞を三次元で培養することにより作製されている。 These Many cell membranes are prepared by culturing cells three dimensional absorbent material derived from a living organism, typified by collagen as a substrate for cell growth. しかしながら、このようにして作製した細胞膜は単層構造であり、増殖された細胞層が積層された形で存在する積層細胞層、特に多種類の増殖され、かつこれら細胞層が接着された積層細胞層を作製することは困難であった。 However, cell membrane produced in this manner is a single layer structure, a stacked layer of cells present in the form of cells grown layers are stacked, particularly many kinds of growth and stacked cells in which these cell layer is adhered it has been difficult to produce a layer.

多種類の細胞が多層構造の積層状態で存在する積層細胞層を作製する方法としては、例えば単層の細胞層を積層する方法が用いられているが(非特許文献1)、この方法では単層の細胞層を連続的に積層する必要があり、多くの手間がかかり効率的でない。 The method of multiple types of cells to produce a laminated cell layers present in a laminated state of a multilayer structure, for example, a method of laminating a cell layer of a single layer is used (Non-patent Document 1), in this way a single must continuously laminating a cell layer of the layer, not efficient takes much labor.

解決しようとする課題は、積層細胞層、特に多種類の細胞層が多層構造の積層状態で存在する積層細胞層を簡単に、かつ効率的に作製することができる培養容器、該容器を使用した培養物と培養方法、ならびに前記培養物を使用した生体組織の再生方法の提供することにある。 Problem to be solved is laminated cell layers, in particular various kinds of cell layers easily stacked cell layers present in a laminated state of the multi-layer structure, and was used culture vessels can be efficiently produced, the container how culture and culture, and to provide a reproducing method of a biological tissue using the culture.
なお、本発明でいう多種類の細胞層が多層構造の積層状態で存在する積層細胞層とは、複数の細胞層が積層状態で存在するものは全て含まれる。 Incidentally, various kinds of cell layers in the present invention is a laminated cell layers present in a laminated state of the multilayer structure include everything in which a plurality of cell layers are present in a stacked state.

本発明の第1は、複数の容器状の培養室が液密に積み重ねられた培養容器であって、前記培養室が該培養室に必要とされる培養液を供給可能な供給口とガス供給口を有し、該培養室の少なくとも一個が培養中に分解可能な材料(以下、この材料を生分解性材料ともいう)で形成された底面部を有するものであることを特徴とする培養容器にある。 The first invention is a culture vessel in which a plurality of container-like culture chamber are stacked in a liquid-tight, capable of supplying the supply port and the gas supply of the culture solution in which the culture chamber is required to the culture chamber has a mouth, said at least one culture chamber degradable material (hereinafter, this material also called biodegradable material) in the culture the culture vessel, characterized in that those having a bottom portion formed with It is in.

本発明の第2は、複数の容器状の培養室が上下方向に液密に積み重ねられた培養容器であって、前記培養室が該培養室に必要とされる培養液を供給可能な培養液供給口とガス供給口を有し、該培養室の少なくとも一個が上下の両面部が存在しない容器状物と該容器状物の上下の両面部を液密に被覆することが可能な生分解性材料で形成されたスペーサーで構成されたものであることを特徴とする培養容器にある。 The of the present invention 2 is a culture vessel in which a plurality of container-like culture chamber are stacked in a fluid-tight manner in the vertical direction, the culture solution capable of supplying the culture solution to the culture chamber are required to the culture chamber It has a supply port and a gas supply port, at least one upper and lower double-sided portion is not present container-like material and container-like material of the upper and lower double-sided portion biodegradability that can be coated in a fluid-tight manner to the said culture chamber in the culture vessel, characterized in that those composed of a spacer formed of a material.

以下、本発明の培養容器を図面に基づいて具体的に説明する。 It will be specifically described below on the basis of the culture vessel of the present invention with reference to the drawings.
1. 1. 培養容器の実施態様1 Embodiment of the culture vessel 1
図1の培養容器は、図2に示す6個の容器状の培養室3〜8が上下方向に液密に積み重ねられて構成されている。 Culture container of Figure 1, six container-like culture chamber 3-8 shown in FIG. 2 is constituted by stacking in a liquid-tight manner in the vertical direction. 各培養室3〜8は、そこに播種される増殖用細胞を培養するに適した培養液および酸素あるいは必要に応じてその他のガス、例えば一酸化炭素、不活性ガス等を供給する供給口2を有する。 Each culture chamber 3-8, other gases suitable culture broth and optionally oxygen or necessary to culture the growth for cells seeded therein, for example carbon monoxide, supply port for supplying an inert gas such as 2 having. 前記供給口2を介して個々の培養室に他の培養室に供給される培養液と混合されることなく供給可能となる。 Wherein through the supply port 2 can be supplied without being mixed with each culture fluid supplied to the other culture chamber to the culture chamber. また、前記供給口2を介して酸素も供給することができる。 The oxygen can also be supplied through the feed opening 2.
培養液と酸素あるいはその他のガスを供給する場合、前記供給口2は同一形状のものであってもよいし、あるいは培養液と酸素を供給する場合は別の異なる形状のものとしてもよい。 When supplying the culture solution and oxygen or other gases, the supply port 2 may be of the same shape, or the case of supplying the culture solution and oxygen may be that of another different shape. したがって、一つの培養室に設けられる供給口2は1個でも良いが、好ましくは2個以上の複数個である。 Thus, the supply port 2 provided on one of the culture chamber may be one, but preferably two or more plural. また、酸素を供給する前記供給口2には除菌フィルターを設けてもよく、かつ前記供給口2にはその開口部を外界と遮断可能な栓部材を装着可能なものであってもよい。 Oxygen may be the supply port 2 is provided with a sterilization filter for supplying, and the supply port 2 may be capable mounted outside and can block stopper member and its opening. また、図1および2に示す培養室3〜8の形状は円筒状のものであるが、該形状は任意の形状のものが採用できる。 The shape of the culture chamber 3-8 shown in FIG. 1 and 2 are those cylindrical, the shape may be adopted are of any shape. 前記のように、本発明の培養容器は、各培養室に播種された細胞毎に好ましい培養液が他の培養液と混合されることなく供給でき、かつ各培養室は好ましい培養条件、例えば酸素供給量、酸素供給速度、培養温度等を独立してコントロールできるので、本発明の培養容器は多種類の増殖された積層細胞層を作製するのに特に好都合である。 As the culture vessel of the present invention, preferably the culture medium every cells seeded in each culture chamber can be supplied without being mixed with other cultures, and the culture chamber is preferred culture conditions, such as oxygen feed rate, oxygen supply rate, so can be controlled independently of the culture temperature, culture vessels of the present invention is particularly advantageous for producing a laminated cell layers of the multi-type growth.

前記培養室3〜8は、その底面部31〜81は培養中に分解可能な生分解性材料で形成され、さらに好ましくは底面部81を構成する生分解性材料が、他の底面部31〜71を構成する生分解性材料に比較して分解速度が低いことが好ましい。 The culture chamber 3-8, the bottom portion 31 to 81 is formed of a degradable biodegradable material in the culture, more preferably biodegradable material constituting the bottom portion 81, the other bottom surface portion 31 to degradation rate compared to the biodegradable material constituting the 71 low it is preferable. 培養室3〜8の底面部31〜81の分解特性を前記のようなものとし、さらにはその厚さを調節することにより、増殖用細胞層の所定の培養が完了した時点で、最下層の培養室以外の増殖用細胞層を載持する底面部を少なくとも部分的、好ましくは完全に破壊するものであるので、前記培養容器は所定の培養が完了した時点では、図4に示すように最下層の培養室以外の培養室で増殖された増殖細胞層は最下位の培養室8の底面部81上に重積あるいは積層されたものとなる。 The decomposition characteristics of the bottom portion 31 to 81 of the culture chamber 3-8 shall the like, more by adjusting its thickness, when the predetermined culturing is complete proliferation for cell layer, the lowest layer of at least partially the bottom section Jisuru mounting proliferation for cell layer other than the culture chamber, so preferably those completely destroy the culture vessel at the time of a given culture is completed, most as shown in FIG. 4 lower proliferating cells layer grown in culture room other than the culture chamber of the those Juseki or stacked on the bottom portion 81 of the lowermost of the culture chamber 8. また、前記増殖細胞層の重積物あるいは積層物は、前記底面部81がそこに載持されている載持物を保持するに十分に強度等の維持しているから、作製された積層あるいは積層細胞層の取扱い、例えば生体へ移植する等に際しての取扱い、あるいは移植の作業が簡単となる。 Further, intussusception thereof, or laminates of the proliferating cells layer, since the bottom surface portion 81 is maintained such sufficiently strength to hold the personal effects placement that is diacetic therein, stacked or laminated was prepared handling of cell layers, for example, handling during such implantation into the living body or the work of transplantation, it is simplified.
前記のようにして得られた重積あるいは積層増殖細胞層は、底面部81と一緒に培養室81から取り外すことにより生体内に移植することができる。 Stacking or laminating proliferation cell layer obtained as described above may be implanted in the body by removing from the culture chamber 81 with the bottom portion 81. また、前記培養室3〜8は底面部を除いて再使用が可能なように培養段階で分解しないものが好ましく、例えばポリスチレンで構成されたものが挙げられる。 Moreover, it said culture chamber 3-8 is preferably one that does not decompose in culture step to allow reuse except the bottom portion, and those which are composed of, for example, polystyrene. ただし、培養室81全体をその底面部81を含めて所定の培養が完了した時点で未分解であるが、生体内では分解可能な材料で作製すれば、所定の培養が完了した時点で底面部81を培養室81から取り外すことなく、前記培養物をそのまま生体内に移植することができる。 However, although the entire culture chamber 81, including its bottom portion 81 is undegraded when given culture is completed, be manufactured in a degradable material in vivo, the bottom portion when the given culture is completed 81 without removing from the culture chamber 81, and the culture can be directly implanted in vivo.

前記培養室3〜8の底面部31〜81は、各培養室に供給される培養液および増殖細胞が混合しないように、液透過性が低いものを用いることが好ましい。 Bottom of the culture chamber 3-8 31-81, as the culture liquid and proliferating cells supplied to each culture chamber is not mixed, it is preferable to use a low liquid permeability. より好ましくは無孔のもの、例えば無孔のフィルムを使用することが好ましい。 More preferably a non-porous, for example, it is preferable to use a film of non-porous. さらに、前記底面部31〜81は、そこに載持される前記増殖用細胞層の該フィルムへの接着性が向上するように、プラズマ照射、コロナ放電等により表面改質したものが好ましく、また、このフィルムの厚さのコントロール及び/又は底面部31〜81を構成する材料を適宜選択することにより、前記底面部31〜81の培養段階での分解速度を任意にコントロールすることができる。 Further, the bottom surface portion 31 to 81, said as adhesion to the film growth for cell layer is improved to be diacetic therein, plasma irradiation is preferably one surface-modified by the corona discharge or the like, also , by selecting the material constituting the thickness of the control and / or bottom portions 31-81 of the film appropriately, it is possible to arbitrarily control the degradation rate of the culture stage of the bottom surface portion 31-81. ただし、前記底面部31〜81を構成する部材はフィルム形状のものに限られるものではなく、前記のような培養室に要求される要件を満足するものであれば、前記増殖用細胞層を載持可能なもの、例えば平面状スポンジあるいは繊維束等のようなものであっても良い。 However, member constituting the bottom surface portion 31 to 81 is not limited to those of the film-shaped, as long as it satisfies the requirements required of the culture chamber as described above, placing the proliferation for cell layer capable lifting, for example may be one such as planar sponge or fiber bundles. また、前記培養室8の底面部81は未分解で生体組織内に移植された場合、再生誘導のための足場としての機能を奏することができる。 Further, the bottom portion 81 of the culture chamber 8 when implanted into living tissue undecomposed can exhibit the function as a scaffold for the regeneration-inducing. すなわち、通常、培養物を生体組織に移植して組織の再生誘導を行う場合、血液細胞を除いて欠損部位組織の再生誘導のために足場を必要とするが、前記最下層の培養棚が前記のような生体組織の再生誘導における足場の機能を奏することにより、別途生体組織の再生誘導に際して別途前記のような足場の機能を奏する材料を使用する必要がなく、生体組織の再生誘導を簡単に行うことができるという効果を奏することができる。 That is, usually, when performing reproduction induction of transplantation to tissue cultures to a living tissue, requires a scaffold for regeneration induction of defect site tissue except blood cells, the lowermost culture shelves the by achieving the function of the scaffold in regeneration-inducing biological tissue, such as, it is not necessary to use a material that exhibits separate the function of the scaffold, such as when separate regeneration-inducing biological tissue, easily reproduced induction of biological tissue it can be an effect that can be performed.
本発明の培養容器における培養室の数および培養室に播種される増殖用細胞の種類は、作製される積層細胞層の使用分野あるいは用途に応じて適宜決定される。 Type of growth for cells seeded on the number and the culture chamber in the culture chamber in the culture vessel of the present invention is appropriately determined depending on the field of use or application of the laminated cell layers to be manufactured.

2. 2. 培養容器の実施態様2 Implementation of the culture vessel aspects 2
図3に基づいて説明する。 It will be described with reference to FIG.
本実施の態様の培養容器は、前記図2に示す培養室3〜8の底面部を有しないものに相当する容器状部材32〜82を各容器状部材間にスペーサー10〜14を挟んで積み重ね、最上位の容器状部材32の上部開口部を蓋部材9で閉鎖し、また最下位の容器状部材82の下部開口部を生分解性材料で形成された蓋部材15で閉鎖して構成されるものである。 The culture vessel of this embodiment of the stacked sandwich the spacer 10 to 14 the container-like member 32-82 corresponding to those having no bottom portion of the culture chamber 3-8 shown in FIG. 2 between the container-like member is configured to close the upper opening of the container-like member 32 of the uppermost closed with a lid member 9, also with a cover member 15 which the lower opening formed by the biodegradable material of the lowest of the container-like member 82 is shall. 蓋部材15を使用する代わりに最下位の容器状部材82のみを前記実施の態様1で使用する有底の培養室8と同様のものを使用してもよく、また、前記蓋部材9は非生分解性材料である必要はなく、任意の材料で形成されたものでよい。 May be used as only the lowest container-like member 82 instead of using the cover member 15 similar to the bottom of the culture chamber 8 for use in aspects 1 of the embodiment, also, the cover member 9 is non need not be biodegradable materials may be those formed of any material.
本実施の態様の培養容器を構成するスペーサーの材料としては、前記実施態様1の培養室の底面部31〜81を構成するものと同様のものが用いられ、また、本実施の態様の培養容器を構成する容器状部材32〜82の材料としては、前記実施態様1の底面部31〜81を除く培養室3〜8を構成するものと同様の材料が用いられる。 The material of the spacer constituting the culture vessel of this embodiment, the used the same components as those constituting the bottom surface portion 31 to 81 of the culture chamber embodiment 1, also culture vessels aspect of this embodiment as the material of the container-like member 32-82 which constitute the, same materials as those constituting the culture chamber 3-8 except the bottom surface portion 31 to 81 of the embodiment 1 is used.
本実施態様の培養容器は、スペーサーを用いることにより、実施態様1と同様の機能を奏することができるだけでなく、使用するスペーサーの種類を簡単に変更することができるので、各培養室に合わせて所望の分解速度のものを容易に得ることが可能である。 Culture vessel of the present embodiment, by using a spacer, not only can achieve the same functions as in embodiment 1, it is possible to easily change the type of spacers used, in accordance with each culture chamber it is possible to easily obtain those desired degradation rate.

前記実施態様1および2の培養容器においては、培養室間がリング状シール部材で液密にシールされていることが好ましい。 Wherein in the culture vessel of embodiments 1 and 2, it is preferable that between the culture chamber is sealed liquid-tightly by a ring-shaped seal member. 前記リング状シール部材としては、弾性を有するリング状物、例えば例えば図に示すようなシリコーンゴムのリング状物が挙げられるが、本発明で使用される前記リング状シール部材はシリコーンゴムのリング状物に限定されるものではなく、前記培養室間を液密にシールできるリング状シール部材であれば特に制限なく使用が可能である。 Examples of the ring-shaped seal member, a ring-shaped material having elasticity, for example, for example, a ring of silicone rubber as shown in FIG. Among them, the ring-like seal member used in the present invention like silicone rubber ring is not limited to objects, in particular it can be used without limitation as long as a ring-shaped seal member can seal in a liquid tight manner between the culture chamber. また、前記シールは他の任意のシール手段、あるいはシール材を使用できる。 Moreover, the seal is any other sealing means or sealing material may be used. このようなシール手段、あるいはシール材としては、例えば培養後には各培養室を簡単に分離できるように培養室の外面から簡単に剥離可能な感圧性粘着テープのようなテープ部材、あるいは接着剤などを挙げることができる。 Such sealing means, or as a sealant, for example, a tape member, such as easy peelable pressure-sensitive adhesive tape from the outer surface of the culture chamber so can easily separate the culture chamber after the culture, or an adhesive such as it can be mentioned. このようなシール手段は、本発明の培養容器の断面形状は円形状のものに限定されるものではなく任意の断面形状、例えば四角形のもののようなものも採用可能であるが、このような形状の培養容器の場合においても、培養室間をシールする際に有効である。 Such sealing means, the sectional shape of the culture vessel of the invention any cross-sectional shape is not limited to a circular shape, for example like a rectangular ones can also be employed, such a shape in each case the culture vessel is effective in sealing between the culture chamber.

前記実施態様1および2の培養容器培養室の底面部、あるいはスペーサーを構成する生分解性材料としては、大きく分けて合成高分子材料、天然高分子材料および無機材料がある。 As the biodegradable material forming the bottom portion of the culture vessel culture chamber of embodiments 1 and 2, or a spacer, synthetic polymer material roughly, natural polymeric materials and inorganic materials.
高分子材料としては、ポリグリコール酸、ポリ乳酸、ポリカプロラクトン、乳酸−グリコール酸共重合体、乳酪−カプロラクトン共重合体等が挙げられ、特に前記共重合体はその共重合成分の共重合比を変えることにより、その特性、例えば前記生分解性の速度を変更することができる。 As the polymer materials, polyglycolic acid, polylactic acid, polycaprolactone, lactic acid - glycolic acid copolymer, Nyuraku - caprolactone copolymer and the like, especially the copolymers copolymerization ratio of the copolymer component by changing its properties, it can be modified, for example the speed of the biodegradable.
天然高分子材料としては、コラーゲン、フィブロネクチン、ゼラチン、キチン、キトサン、ヒアルロン酸、アルギン酸等が挙げられる。 The natural polymeric materials, collagen, fibronectin, gelatin, chitin, chitosan, hyaluronic acid, alginic acid and the like. また、これら天然高分子材料、コラーゲン、ゼラチン等は架橋処理することにより、その生分解性を任意に変更することができる。 These natural polymeric materials, collagen, by gelatin is to crosslinking treatment, it is possible to arbitrarily change its biodegradability. その他、生分解性無機材料としては、リン酸三カルシウム、炭酸カルシウムが挙げられる。 Examples of other biodegradable inorganic materials, tricalcium phosphate, calcium carbonate.

本発明の培養容器を使用した増殖用細胞の培養は、例えば以下のようにして行うことができる。 Culture growth for cells using culture vessels of the present invention can be performed, for example, as follows.
本発明の前記培養容器の培養室に、作製した細胞層自体の使用分野あるいは用途に応じて適当な増殖用細胞を播種した後、培養液(例えば血清を含有した培養液)を供給して培養を開始し増殖用細胞層を培養し、培養養開始後、最下層の底面部あるいは最下層のスペーサーを除いた底面部あるいはスペーサーが分解消失させ、この分解除去された底面部あるいはスペーサーに載持された増殖用細胞層を下層の増殖用細胞層に重力落下させ、前記殖用細胞層が接着された積層細胞層を形成させる。 The culture chamber of the culture vessel of the present invention, after seeding the appropriate growth for cells depending on the field of use or application of the produced cell layer itself, by supplying a culture solution (e.g., serum containing culture medium) Culture start by culturing proliferating a cell layer, after culturing nutrient initiation, bottom portion or spacer excluding the bottom portion or the lowermost spacer of the lowermost allowed is decomposed disappearance diacetic this decomposition removed bottom portion or spacer has been the proliferation for cell layer is gravity in the lower layer of growth for cell layer, the 殖用 cell layer to form a laminated cell layer adhered. 前記のようにして作成した培養物は、それを生体に移植して細胞増殖させることにより生体組織の再生を行うことができる。 Cultures were prepared as described above, it is possible to reproduce the living body tissue by transplanted into a living body to cell proliferation.

多種類の細胞層が層状構造に積層した培養容器および該培養容器を使用した細胞膜の作製方法 The method for manufacturing a membrane using a wide variety of culture vessels and the culture container cell layers are stacked in a layered structure

生分解性材料で形成された底面部を有する培養室の複数個が積み重ねられて構成された培養容器 図1および2に基づいて説明する。 Will be described with reference to the culture vessel Figure 1 and 2 constituted by a plurality are stacked culture chamber having a bottom portion formed of a biodegradable material.
分子量10万のポリグリコール酸を用いて押し出し成形により厚みが200μmのフィルム、および同様にして乳酸−カプロラクトン共重合体のフィルムを得た。 Thickness by extrusion molding using a polyglycolic acid having a molecular weight of 100,000 films of 200 [mu] m, and similarly to lactic acid - to obtain a film having a caprolactone copolymer. 得られた前記両フィルムは放電処理により親水化処理して増殖用細胞層に対する接着性を改善し、前記ポリグリコール酸フィルムを上下両面部が無く、かつ、その側壁面部に培養液および酸素を供給する開口部2を有するポリスチレン製容器状物の一方開口部面に貼り付けて5個の培養室3〜7を作製し、また、前記前記ポリグリコール酸フィルムに比較して生分解速度の遅い乳酸−カプロラクトン共重合体のフィルムを上下両面部が無いポリスチレン製容器状物の一方開口部面に貼り付けて1個の培養室8を作製した。 The resulting wherein both film is hydrophilized to improve the adhesion to the growth for cell layer by the discharge treatment, the polyglycolic acid film without upper and lower surfaces portions, and the culture solution and supplying oxygen to the sidewall portions to produce one opening surface adhered five culture chamber 3-7 polystyrene container-like material having an opening 2 which, also, the said polyglycolic acid film slow to the rate of biodegradation compared to lactic acid - paste film caprolactone copolymer on one opening surface of the upper and lower surfaces portions is no polystyrene container-like material was produced one culture chamber 8. 前記培養室8を最下位として、前記培養室3〜8を積み重ね6層の培養室よりなる培養容器を作製し、γ線滅菌した。 The culture chamber 8 as the least significant, to produce a culture vessel made of culture chamber six layer stack the cultivating chamber 3-8, and γ-ray sterilization.

生分解性材料で形成されたスペーサーを用いて構成された培養容器 図3に基づいて説明する。 It will be described with reference to the culture vessel Figure 3 constructed using a spacer formed of a biodegradable material.
分子量10万のポリグリコール酸を用いて押し出し成形にて厚みが200μmのフィルム、および同様にして乳酸−カプロラクトン共重合体のフィルムを得た。 The thickness at extrusion using a polyglycolic acid having a molecular weight of 100,000 films of 200 [mu] m, and similarly to lactic acid - to obtain a film having a caprolactone copolymer. 前記両フィルムは放電処理により親水化処理して増殖用細胞層に対する接着性を改善した。 The two films were improved adhesion to hydrophilic treatment to grow for cell layer by the discharge treatment. 上下両面部が無く、かつその側壁面部に培養液および酸素を供給する開口部2を有するポリスチレン製容器状物32〜82の6個を、各容器状物間に前記ポリグリコール酸フィルムを挟んで積み重ねるとともに、前記ポリスチレン製容器状物82の下部開口部面を前記前記ポリグリコール酸フィルムに比較して生分解速度の遅い乳酸−カプロラクトン共重合体のフィルムで液密にシールして培養容器を作製し、γ線滅菌した。 No upper and lower surfaces unit, and six polystyrene container-like product 32 to 82 having an opening 2 for supplying the culture liquid and oxygen to the sidewall portions, sandwiching the polyglycolic acid film between each container-like material with stacking, the lower opening surface wherein the polyglycolic acid film in comparison to biodegradation slow lactate of the polystyrene container-like material 82 - Preparation of the culture vessel and liquid-tightly sealed with a film of caprolactone copolymer and, it was γ-ray sterilization.
なお、前記実施例1および2の培養容器においては、各培養室間は帯状のシリコーンゴムのリング状物、あるいは感圧接着テープのようなテープ等によって液密にシールされている。 Incidentally, in the above culture vessel of Example 1 and 2, between each culture chamber is liquid-tightly sealed by tape or the like, such as a band-shaped ring of a silicone rubber or pressure sensitive tape.

前記実施例1の培養容器を使用した重積あるいは積層細胞層の作製方法 図1に基づいて説明する 図1の培養容器において、培養室が3層のものを使用した。 In culture vessel of FIG. 1 will be described with reference to manufacturing process diagrams 1 of intussusception or stacked cell layers using culture vessels of the Example 1, the culture chamber was used a three-layer. 最上位の底面部がポリグリコール酸フィルムで構成される培養室(第1の培養室)に表皮細胞、中間の底面部がポリグリコール酸フィルムで構成される培養室(第2の培養室)に真皮細胞、最下位の底面部が乳酸−カプロラクトン共重合体フィルムで構成される培養室(第3の培養室)に血管内皮前駆細胞を、別々に1.0×10 4個を播種した。 Epidermal cells in the culture chamber formed bottom portion of the uppermost polyglycolic acid film (first culture chamber), the culture chamber comprised bottom of intermediate polyglycolic acid film (the second culture chamber) dermal cells, the lowest bottom part lactic acid - caprolactone copolymer film culture chamber consisting of vascular endothelial progenitor cells (third culture chamber) were seeded separately 1.0 × 10 4 pieces of. 各培養室に供給口2を介して増殖対象の前記表皮細胞、真皮細胞および血管内皮前駆細胞の培養に適した培養液を入れた後、前記供給口2を介して酸素を供給して培養することで細胞を増殖させ、培養開始から20日後、第1と第2の培養室の底面部を構成するポリグリコール酸フィルムが完全に消失して、かつ前記第1と第2の培養室で増殖された増殖細胞層は、最下位の培養室(第3の培養室)の乳酸−カプロラクトン共重合体のフィルムで構成される底面部上に落下して前記各増殖細胞層問が接着した多層構造の積層あるいは重責した細胞層を得た。 The epidermal cell proliferation subject through the supply port 2 in each culture chamber, was placed in a culture medium suitable for culturing dermal cells and vascular endothelial progenitor cells, cultured by supplying oxygen through the supply port 2 cells were grown by, after 20 days from the initiation of culture, grown in the first and in the polyglycolic acid film constituting a bottom portion of the second culture chamber completely disappeared, and the first and second culture chamber have been grown cell layer bottom of the culture chamber (third culture room) lactic acid - multilayer structure caprolactone copolymer of the respective proliferating cells layers Q to fall onto the formed bottom portion of a film is adhered It was obtained in laminated or burden cell layer.
本実施例の積層細胞層の作製方法によると、各培養室の増殖細胞の培養を各培養細胞の培養に適した培養液をそれぞれ別個に供給して培養を行うことができ、かつ各培養室の培養条件をコントロールすることが可能であるので、多層に重積あるいは積層した細胞層層を製造する方法として非常に有効である。 According to the manufacturing method of the laminated cell layers of the present embodiment, it is possible to carry out the culture broth suitable for culturing the cultured cells cultured in proliferative cells in each culture chamber separately supplied, and each culture chamber since it is possible to control the culture conditions, it is very effective as a method for producing a Juseki or stacked cell layer layer in a multilayer.
なお、前記表皮細胞、真皮細胞および血管内皮前駆細胞の培養に適した培養液としては、それぞれ従来用いられているものを使用することができる。 Incidentally, the skin cells, the culture medium suitable for culturing dermal cells and vascular endothelial progenitor cells, may be used those conventionally used, respectively.

本発明の培養容器を利用して、例えば皮膚再生用の積層細胞層層を製造することができる。 Using the culture container of the present invention can be produced, for example, stacked cell layer layer for skin regeneration. すなわち、皮膚は角化細胞、色素細胞やメルケル細胞等で構成される表皮層、線維芽細胞と呼ばれる細胞でつくられた線維と基質で構成される真皮層および真皮の下にあり、脂肪細胞を含み、血管や神経幹も走っている下皮を基本構成とするものであるが、皮膚細胞は他の生体組織、またこれに代わる物質に付着して増殖し,平面上に層を作る性質があり、従来、細胞培養床としてポリスチレンや合成高分子物質に天然高分子物質をコーティングしたもの等を利用して細胞増殖が行われている。 That is, skin keratinocytes, the epidermal layer composed of pigment cells and Merkel cells, etc., dermal layer and composed of fibers and substrates made of cells called fibroblasts located below the dermis, adipocytes wherein, while the hypodermis, also run blood vessels and neural stem in which the basic structure, skin cells grow attached other biological tissue and the material in place of this, the property of making the layers on a plane There, conventionally, a polystyrene and a synthetic polymer material by using those coated with natural polymer substances such as cell growth has been performed as a cell culture bed.
本発明の培養容器を利用すると、皮膚再生用の積層細胞層の他、血管再生用の積層細胞層、内臓臓器再生用の積層細胞層等を容易に作成することができる。 Utilizing culture vessel of the present invention, other laminated cell layers for skin regeneration, laminated cell layers for revascularization, the laminated cell layers for visceral organ regeneration, etc. can be easily created.

実施例1の培養容器の構成を示した説明図である。 It is an explanatory view showing a configuration of the culture vessel of Example 1. 実施例1の培養容器の培養室の構成を示した説明図である。 It is an explanatory view showing a configuration of the culture chamber in the culture vessel of Example 1. 実施例2の培養容器の構成部材を示した説明図である。 Is an explanatory view showing the components of the culture vessel of Example 2. 実施例3の培養が終了した時点における、培養容器の状態を示した説明図である。 At the time the culture is terminated in Example 3 is an explanatory view showing a state of the culture vessel.

符号の説明 DESCRIPTION OF SYMBOLS

1 培養容器の蓋 2 培養液あるいは酸素ガス供給口 3 生分解性材料の底部を有する円筒容器 4 生分解性材料の底部を有する円筒容器 5 生分解性材料の底部を有する円筒容器 6 生分解性材料の底部を有する円筒容器 7 生分解性材料の底部を有する円筒容器 8 生分解性材料の底部を有する円筒容器 31 生分解性材料の底部 41 生分解性材料の底部 51 生分解性材料の底部 61 生分解性材料の底部 71 生分解性材料の底部 81 生分解性材料の底部 32 上下両面部の無い円筒状部材 42 上下両面部の無い円筒状部材 52 上下両面部の無い円筒状部材 62 上下両面部の無い円筒状部材 72 上下両面部の無い円筒状部材 82 上下両面部の無い円筒状部材 9 培養容器の蓋 10 生分解性材料で形成された円盤状スペーサー 11 生 Cylindrical container 6 biodegradable having a bottom of the cylindrical container 5 biodegradable material having a bottom of the cylindrical container 4 biodegradable material having a bottom portion of the lid 2 culture or oxygen gas supply port 3 biodegradable material of the culture vessel the bottom of the bottom 51 biodegradable material of the bottom 41 biodegradable material of the cylindrical container 31 biodegradable material having a bottom of the cylindrical container 8 biodegradable material having a bottom of the cylindrical container 7 biodegradable material having a bottom material 61 biodegradable bottom bottom 81 biodegradable bottom 32 upper and lower surfaces portion without the cylindrical member 42 upper and lower surfaces portion without the cylindrical member 52 upper and lower surfaces portion without the cylindrical member 62 and below the material 71 biodegradable material material sided portion without the cylindrical member 72 upper and lower surfaces portion without the cylindrical member 82 upper and lower surfaces portion without the cylindrical member 9 discotic spacer 11 raw formed by the lid 10 biodegradable material of the culture vessel 解性材料で形成された円盤状スペーサー 12 生分解性材料で形成された円盤状スペーサー 13 生分解性材料で形成された円盤状スペーサー 14 生分解性材料で形成された円盤状スペーサー 15 生分解性材料で形成された蓋 A 増殖した表皮細胞層 B 増殖した真皮細胞層 C 増殖した血管内皮前駆細胞層 Discoid spacer 15 biodegradable formed in a disk shape spacer 14 biodegradable material formed in a disk shape spacer 13 biodegradable material formed in a disk shape spacer 12 biodegradable material formed by disintegratable material endothelial progenitor cells layer dermal cell layers were C proliferation in epidermal cell layer B proliferation in cover A growth formed of a material

Claims (6)

  1. 複数の容器状の培養室が上下方向に液密に積み重ねられて構成され、下記要件(1)〜(4)を満足するものであることを特徴とする培養容器。 A plurality of container-like culture chamber is constituted by stacking in a liquid-tight manner in the vertical direction, the culture vessel, characterized in that those satisfying the following requirements (1) to (4).
    (1)培養室が該培養室に必要とされる培養液を供給可能な供給口とガス供給口を有すること (1) having a supply port and a gas supply port capable of supplying the culture liquid culture chamber is required to the culture chamber.
    (2)培養室の底面部が生分解性材料で形成されること (2) a bottom portion of the culture chamber Rukoto formed of a biodegradable material.
    (3) 最下位の培養室を除く培養室の底面部は培養が完了した時点で分解して該底面部に形成された培養物を容器の下方に重力落下させることが可能な生分解性材料で構成されること。 (3) lowest decompose biodegradable material capable of gravity cultures formed bottom surface below the container bottom portion of the culture chamber when the culture is completed, except for the culture chamber of in be configured.
    (4) 最下位の培養室の底面部は培養が完了した時点で前記重力落下させた培養物を保持するに十分な強度を有するものであること。 (4) It is the bottom portion of the culture chamber of the lowest has a sufficient strength to hold the culture was the gravity when the culture is completed.
  2. 複数の容器状の培養室が上下方向に液密に積み重ねられて構成され、下記要件(1)〜(4)を満足するものであることを特徴とする培養容器。 A plurality of container-like culture chamber is constituted by stacking in a liquid-tight manner in the vertical direction, the culture vessel, characterized in that those satisfying the following requirements (1) to (4).
    (1)培養室が該培養室に必要とされる培養液を供給可能な供給口とガス供給口を有すること (1) having a supply port and a gas supply port capable of supplying the culture liquid culture chamber is required to the culture chamber.
    (2)培養室が上下の両面部が存在しない容器状物で、該容器状物間に該容器状物の上下の両面部を液密に被覆するスペーサーを有すること (2) in a container-like material culture chamber is not present both sides of the vertical, having a spacer for covering in a liquid-tight manner on both sides of the upper and lower container-like material between the container-like product.
    (3) 前記スペーサーは培養が完了した時点で分解して該スペーサー面上に形成された培養物を容器の下方に重力落下させることが可能な生分解性材料で構成されたものである (3) the spacer is one which is composed of a biodegradable material capable of gravity cultures formed on the spacer surface is decomposed at the time the culture is completed below the container
    こと。 about.
    (4) 最下位の培養室の底面部は培養が完了した時点で前記重力落下させた培養物を保持するに十分な強度を有する生分解性材料で構成された蓋部材であること。 (4) It is the bottom portion of the culture chamber of the least significant a lid member made of a biodegradable material having sufficient strength to hold the culture was the gravity when the culture is completed.
  3. 培養室間が液密にリング状シール材でシールされていることを特徴とする請求項1または2に記載の培養容器。 Culture container according to claim 1 or 2 between the culture chamber, characterized in that it is sealed with a ring-shaped sealing member in a liquid-tight manner.
  4. 生体組織の再生誘導のために使用されるものであることを特徴とする請求項1〜3のいずれかに記載の培養容器。 Culture container according to claim 1, characterized in that what is used for the regeneration-inducing biological tissue.
  5. 増殖用細胞を各培養室に播種した請求項1〜4のいずれかに記載の培養容器。 Culture vessel according proliferation for cell to any one of claims 1 to 4 were inoculated into each culture chamber.
  6. 請求項1〜5のいずれかに記載の培養容器の培養室中に増殖用細胞を播種後、前記培養室の培養液の供給口より各増殖用細胞の培養に適した培養液の供給を行い、増殖用細胞の培養と最下位に存在する培養室の底面部を除く培養室の底面部の分解を行い 、この結果、 分解された培養室の底面部に載持された増殖用細胞層を最下位の培養室の底面部上に重力落下させるとともに、これら増殖用細胞層を接着させることを特徴とする培養物の作製方法。 After seeding growth for cells in culture chamber in the culture vessel according to claim 1, carried out the supply of the culture medium suitable for culturing the growth for cells from the supply port of the culture medium of said culture chamber performs decomposition of the bottom surface of the culture chamber except for the bottom portion of the culture chamber to be present in the culture and the lowest growth for cells, as a result, growth for cell layers diacetic on the bottom portion of the degraded culture chamber together to gravity to the lowest of the culture chamber of the bottom part on the method of preparing a culture which is characterized by adhering these proliferation for cell layer.
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