JP4529410B2 - The method for manufacturing a culture carrier, cultures and cultures using the culture carrier - Google Patents

The method for manufacturing a culture carrier, cultures and cultures using the culture carrier Download PDF

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JP4529410B2
JP4529410B2 JP2003362950A JP2003362950A JP4529410B2 JP 4529410 B2 JP4529410 B2 JP 4529410B2 JP 2003362950 A JP2003362950 A JP 2003362950A JP 2003362950 A JP2003362950 A JP 2003362950A JP 4529410 B2 JP4529410 B2 JP 4529410B2
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porous layer
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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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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担体、増殖用細胞が播種された前記の培養担体、増殖用細胞が播種された前記の培養担体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 plated laminated cell layers cell layers are stacked in the form of a multilayer, especially many kinds of culture carrier cell layer is used to produce laminated cell layers laminated in the form of multilayer, growth for cells has been said culture carrier, culture and how culture growth for the cultivation carrier 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、特に多種類の細胞が増殖され、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 layer are laminated, grown especially various types of cells, and these cell layers are bonded it is difficult to prepare a laminated cell layers.

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担体、該培養担体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, especially many kinds of simple stacked cell layers present in a laminated state of the cell layer is a multilayer structure, and efficient culture carrier which can be made, using the culture carrier culture and the culture method, 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、非多孔質層と多孔質層を有し、かつ生分解性材料で構成されたものであることを特徴とする細胞培養担体にある。 The first of the present invention has a non-porous layer and a porous layer, and in cell culture carrier which is characterized in that those composed of a biodegradable material.

本発明の第2は、前記第1の細胞培養担体の複数個が多孔質層を上側として積み重ねて構成され、かつ最下層の非多孔質層の生分解速度が、他の非多孔質層や多孔質層の生分解速度に比較して小さいことを特徴とする細胞培養担体にある。 The second of the present invention, a plurality of the first cell culture carrier is configured by stacking the porous layer as the upper, and the rate of biodegradation of the lowermost non-porous layer, Ya other non-porous layer lying in the cell culture carrier, wherein small compared to the rate of biodegradation of the porous layer.

本発明の第3は、前記第1または第2の培養担体の多孔質層中に増殖用細胞培養が播種されていることを特徴とする細胞培養担体にある。 The third of the present invention is a cell culture carrier which is characterized in that the growth cell culture are seeded on the porous layer of the first or second culture carrier.

本発明の第4は、前記第3の細胞培養担体を培養した培養物にある。 The fourth of the present invention is to fermentation by cultivating the third carrier for cell culture.

本発明の第5は、前記第1あるいは第2の細胞培養担体の多孔質層中に増殖用細胞を播種後、該細胞培養担体を積み重ねた状態で増殖用細胞の培養を行うとともに最下位に存在する非多孔質層を除く非多孔質層と多孔質層の分解を行い、培養された増殖用細胞層を未分解の非多孔質層上に重力落下させることを特徴とする増殖用細胞の培養物の作製方法にある。 The fifth of the present invention, after seeding the growth for cells in the porous layer of the first or second carrier for cell culture, the lowest performs culture growth for cells in the state in which stacked the cell culture carrier performs decomposition of non-porous layer and a porous layer other than the non-porous layer which is present, the growth for cells, characterized in that to gravity in proliferation for cell layer cultured undecomposed nonporous layer in the manufacturing method of the culture.

本発明の第6は、前記第4の培養物を生体に移植して細胞増殖させることを特徴とする生体組織の再生方法にある。 Sixth present invention is the fourth cultures reproducing method of the biological tissue, characterized in that to be implanted in a living body is cellular proliferation.

以下、本発明の実施態様を図面に基づいて具体的に説明する。 It will be specifically described below based on an embodiment of the present invention with reference to the drawings.
1. 1. 細胞培養担体の構成本実施態様の図2で示される細胞培養担体は、図1に示される多孔質層1と非多孔質層2を有し、生分解性材料で構成された培養担体およびこの培養担体と同様に多孔質層3と非多孔質層4を有し、生分解性材料で構成されている細胞培養担体の2個をそれぞれ多孔質層を上側として積み重ねたものである。 Carrier for cell culture shown in FIG. 2 of the construction this embodiment of the carrier for cell culture, has a porous layer 1 and the non-porous layer 2 shown in FIG. 1, the culture carrier composed of a biodegradable material and this the porous layer 3 similar to the culture support having a non-porous layer 4, in which each porous layer two carrier for cell culture which is composed of a biodegradable material stack as the upper. 本実施態様の細胞培養担体は上述のように2個の細胞培養担体を積み重ねたものであるが、本発明の培養担体の積み重ね数は目的とす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、例えば再生皮膚用の増殖細胞層を目的とする場合には、 表皮細胞を播種した第1の培養担体、該第1の培養担体の上に真皮細胞を播種した第2の培養担体が積み重ねられ、さらに該第2の培養担体の上に血管内皮前駆細胞皮細胞を播種した第3の培養担体が積み重ねられた3個の細胞培養担体で構成される。 Although the carrier for cell culture of the present embodiment is a stack of two cell culture carrier as described above, the stacking number of the culture carrier of the present invention is appropriately depending on the field of use or application of the laminated cell layers of interest is determined, for example, if it is intended to grow cell layers for reproducing skin first culture carrier seeded with epidermal cells, the second culture carrier is seeded with dermal cells on the culture carrier of the first stacked, further comprised of three cell culture carrier third culture supports are stacked seeded with endothelial progenitor cells epithelial cells on the culture carrier of the second.

前記図2に示す細胞培養担体の多孔質層1と多孔質層3および非多孔質層2と非多孔質層4の生分解速度は、使用する生分解性材料の選択および/または多孔質層の孔形状あるいは多孔度によってコントロールすることができる。 Biodegradation rate of the porous layer 1 and the porous layer 3 and the non-porous layer 2 and the non-porous layer 4 of carrier for cell culture shown in FIG. 2, the selection and / or porous layer of the biodegradable material used it can be controlled by the pore geometry or porosity. すなわち、多孔質層は非多孔質層に比較して生分解速度が速く、また多孔度が大きい程、生分解速度が速く、また多孔質層の多孔形状として、例えば連続多孔層の方が独立多孔気泡層に比較して生分解速度が速く、かつ栄養が供給され易い。 That is, the porous layer has a high biodegradation rate compared to the non-porous layer, also the larger porosity, fast biodegradation rate, and as a porous shape of the porous layer, for example, independently towards the continuous porous layer compared to the porous foam layer fast biodegradation rate, and nutrient is supplied easily.
図2に示す本実施態様の細胞培養担体は、上述のように使用する生分解性材料の選択および/または多孔質層の孔形状あるいは多孔度をコントロールすることによって、最下層の非多孔質層4の生分解速度は他の非多孔質層2や多孔質層1,3の生分解速度に比較してもっとも小さく構成され所定の培養が完了した未分解であるのに対して、多孔質層1,3は生分解速度がもっとも大きく構成され所定の培養が完了する迄に分解する分解速度のものであり、さらに非多孔質層2は生分解速度が前記最下層の非多孔質層4と多孔質層1,3の間の大きさで、該層の上下層として存在する増殖用細胞が播種されている多孔質層1,3が所定の培養が完了する迄は前記多孔質層1,3の増殖用細胞が混合しないように分離層としての機能を奏するが The carrier for cell culture of the present embodiment shown in FIG. 2, by controlling the pore geometry or porosity of the selection and / or porous layer of the biodegradable material used as described above, non-porous layer of the lowermost layer biodegradation rate of 4 is undecomposed given culture is the smallest structure in comparison with the rate of biodegradation other nonporous layer 2 and porous layer 1 and 3 is completed whereas, the porous layer 1,3 are of up to degrade degradation rate completes largest constructed given culture biodegradation rate, further non-porous layer 2 and the non-porous layer 4 of the bottom layer biodegradation rate the size between the porous layers 1 and 3, until the porous layer 1, 3 for proliferation cells present as the layer of the upper and lower layers are seeded to a given culture is completed the porous layer 1, Although 3 of growth for cells exhibit the function as a separation layer so as not to mix 所定の培養が完了した時点で分解するものである。 It is to decompose when given culture is completed.

前記図2に示す細胞培養担体は、前記のような構成を採用することにより、以下のような優れた効果を奏することができる。 Carrier for cell culture shown in FIG. 2, by adopting the like arrangement, it is possible to obtain the following excellent effects.
すなわち、所定の培養が完了した時点においては非多孔質層2や多孔質層1,3は生分解により分解させ、多孔質層1,3において培養された培養物は最下位の非多孔質層4に載持させ、かつ該非多孔質層4を所定の培養が完了した時点においても該非多孔質層4上に載持された培養物を保持するに十分な強度を維持しているので、該培養物の取扱い、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だけでなく、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、前記最下位の非多孔質層4は、生体組織の再生誘導における足場の機能を奏することができるので、別途生体組織の再生誘導に際して前記のような足場の機能を That is, in the time of a given culture is completed nonporous layer 2 and porous layer 1,3 is decomposed by biodegradation, cultures cultured in the porous layer 1 and 3 the lowest non-porous layer 4 is diacetic, and so maintains a sufficient strength to hold the cultures diacetic on nonporous layer 4 even when the non-porous layer 4 a given culture is completed, the except handling of the cultures, for example, handling during such implantation into a living body, or not only the work of transplantation is simplified, usually, when performing reproduction induction of transplantation to tissue cultures living tissue, blood cells It requires scaffolding for regeneration induction of defect sites tissue Te, but the non-porous layer 4 of the lowest, since it is possible to achieve the function of the scaffold in regeneration-inducing biological tissue, a separate regeneration-inducing biological tissue the function of the scaffold, such as when 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。 It is not necessary to use a material that can achieve the effect that it is possible to easily perform playback induction of biological tissue.
なお、本実施態様の細胞培養担体においては、細胞培養担体の円柱形状のものを採用しているが、円柱形状のものに限らず、非多孔質層および多孔質層を形成できるものであれば任意の形状のもの、例えば4角形や膜形状のものが挙げられる。 In the carrier for cell culture of the present embodiment adopts the one cylindrical carrier for cell culture, not limited to a cylindrical shape, as long as the non-porous layer and porous layer can be formed of any shape, it can be mentioned, for example, square or film shape.

本実施態様を含めて本発明で採用可能な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。 Biodegradable materials which can be employed in the present, including the present embodiment invention, the 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.

2. 2. 細胞培養担体の作製法 本発明の培養担体の作製方法としては、例えば以下のようなものが挙げられる。 As a method for manufacturing a culture carrier of production method invention of the cell culture carriers include for example, as follows.
(1)多孔質体の一部を溶剤処理または熱処理して非多孔質化する方法(2)非多孔質体の一部を凍結乾燥処理して多孔質化する方法(3)多孔質層層と非多孔質層を接合する方法 前記(1)の方法で使用する多孔質体としては、例えば生分解性材料を発泡剤を使用して発泡させた発泡体が挙げられ、この発泡体の発泡条件あるいは使用する発泡剤の種類を選択することにより、適宜その多孔度および/または孔形状をコントロールすることができる。 (1) a method of a part of the porous body made porous by freeze drying a portion of the solvent treatment or heat treatment to non-porous to the method (2) non-porous body (3) the porous layer layer and a porous material used in the method of the method for bonding the non-porous layer (1), for example a biodegradable material was foamed using a foaming agent foams are exemplified, foaming of the foam by selecting the type of blowing agent condition or use, it can be appropriately controlled and the porosity and / or pore geometry. また、多孔質層における孔の大きさは、50〜500μmが好ましい。 The size of the pores in the porous layer, 50 to 500 [mu] m is preferred. 多孔質層における孔の大きさが50未満では、細胞・培養液が入りにくくなり、十分に細胞が育たない。 The size of the pores in the porous layer is less than 50, less likely to enter the cell-culture medium, it is not grow sufficiently cells. 500μmを超えると強度が弱くなる。 Intensity is weaker than the 500 [mu] m.

3. 3. 前記細胞培養担体を利用した増殖用細胞の培養方法 目的の増殖細胞層の層数に応じた数の前記細胞培養担体を準備し、各細胞培養担体の多孔質層の孔中に目的の増殖細胞を得るために必要な増殖用細胞を播種した。 Prepare the number the carrier for cell culture according to the number of layers of proliferation cell layer culture method object of proliferation for cells using the cell culture support, the purpose of proliferating cells in the pores of the porous layer of the carrier for cell culture They were seeded growth for cells required to obtain the. この増殖用細胞の播種は、例えば前記増殖用細胞を滅菌、例えばγ線滅菌した前記細胞培養担体の多孔質層面上に供給した後、該供給された増殖用細胞を多孔質層中に埋入させることにより行うことができる。 Embedded seeding the growth for cells, for example, by supplying the growth for cells sterilization, for example, the porous layer surface on the carrier for cell culture was γ ray sterilization, the growth for cells the feed to the porous layer it can be carried out by. 前記埋入手段としては、例えば前記増殖用細胞を播種した細胞培養担体を遠心させる遠心回転手段、あるいは滅菌された圧力ガスによる圧入手段が挙げられる。 As the implantation means, for example a centrifugal rotating means is a centrifugal cell culture carrier seeded with the proliferation for cell, or press-fit means and the like through sterile pressure gas.
また、前記増殖用細胞は前記埋入手段によっては多孔質層の表面部分にしか埋入できない場合があるとしても、生分解性材料の分解に伴い多孔質層内部にも埋入、あるいは増殖が可能となる。 Further, the even growth for cells by the implantation device may not be able to enter embedded only in the surface portion of the porous layer, biodegradable due to degradation of the material porous layer inside implanted even or proliferation, It can become.

上述のようにして増殖用細胞を播種した細胞培養担体を必要な個数だけ積み重ね、該積み重ね状態で培養液(例えば血清を含有した培養液)を有する容器中に浸漬して増殖用細胞層を培養し、培養養開始後、最下層の非多孔質層を除いた非多孔質層非および多孔質層が分解消失させ、前記多孔質層中で増殖された培養細胞層を最下層の非多孔質層上に重力落下させ積層細胞層を形成させる。 Stacking a required number of seeded cell culture carrier proliferation for cell as described above, the culture proliferation for cell layer was immersed in a container with a culture solution in the stacked state (eg, serum containing culture medium) and, after culturing nutrient initiation, non-porous layer non and the porous layer, excluding the non-porous layer of the lowermost layer so that decompose loss, nonporous bottom layer of cultured cell layer grown in the porous layer is gravity on the layer to form a laminated cell layers. 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.

細胞培養担体および該培養担体を使用した細胞膜の作製方法 The method for manufacturing a cell membrane using cell culture carriers and the culture supports

細胞培養担体1および該細胞培養担体を使用した積層細胞層の作製方法 図1および2に基づいて説明する。 It will be described with reference to the manufacturing method Figure 1 and 2 of the cell culture carrier 1 and a laminated cell layer using the carrier for cell culture.
分子量10万の乳酸−カプロラクトン共重合体を1,4−ジオキサンに溶解し5%の溶液とし、この溶液を膜状で−50℃にて凍結した。 Lactic acid having a molecular weight of 100,000 - caprolactone copolymer was dissolved in 1,4-dioxane and 5% of the solution was frozen at -50 ° C. The solution is film-like. この膜状凍結物の片面のみを25℃に2分置き溶解させた後に、凍結乾燥法を用いて片面のみが多孔質化した3個の乳酸−カプロラクトン共重合体膜(厚さ500μm)を細胞培養担体として得た。 Only one surface of the film-like frozen material in After every 2 minutes dissolved in 25 ° C., 3 pieces of lactic acid only one side using a freeze-drying method is porous - caprolactone copolymer film (thickness 500 [mu] m) cells It was obtained as a culture carrier. 得られた前記乳酸−カプロラクトン共重合体膜の少なくとも多孔質面は放電により親水化処理を行って細胞接着性を向上させた。 It obtained the lactic - at least the porous surface of the caprolactone copolymer film by performing a hydrophilic treatment to improve cell adhesion by the discharge. さらにこの細胞培養担体の少なくとも多孔質層面、好ましくは全体をγ線滅菌した。 The least porous layer surface of the cell culture support and preferably sterilized whole γ-rays further. 上述のように親水化処理およびγ線滅菌を行った3個の細胞培養担体の多孔質面上に、それぞれ表皮細胞、真皮細胞、血管内皮前駆細胞をそれぞれ1.0×10 個ずつ播種し、播種後、300rpmで遠心することで多孔質内部細胞を埋入した。 On a porous surface of the three cell culture carrier subjected to hydrophilic treatment and γ-ray sterilization as described above, epidermal cells, respectively, were seeded dermal cells, vascular endothelial progenitor cells by 1.0 × 10 4 cells respectively after seeding, it was embedded a porous inner cell by centrifugation at 300 rpm. 前記増殖用細胞を播種した膜状の細胞培養担体を積層して、該積層状態で10%血清含有培養液中に浸漬して培養を行った。 By laminating a film-like carrier for cell culture seeded with the growth for cells, immersed in the culture was performed in 10% serum-containing culture medium in the laminated state. 15日後には多孔質部位はほぼ分解し、消失部分に細胞が増殖されていることが確認された。 After 15 days porous site is almost decomposed, that cells have been grown was confirmed to disappear moiety. 培養開始から20日後、非多孔質層が分解され始め、各細胞膜の接着が見られた。 After 20 days from the initiation of culture, non-porous layer begins to be decomposed, adhesion of the cell membrane was observed.

細胞培養担体2および該細胞培養担体を使用した積層細胞層の作製方法 前記実施例1において、前記表皮細胞および真皮細胞を播種する細胞培養担体は、分子量10万の乳酸−カプロラクトン共重合体をポリグリコール酸に替えた以外は同様にして作製し、また、血管内皮前駆細胞を播種する細胞培養担体は実施例1と同様にして作製した。 In the preparation method of Example 1 of the cell culture carrier 2 and the laminated cell layers using the carrier for cell culture, cell culture carrier for sowing the epidermal cells and dermal cells include lactic acid having a molecular weight of 100,000 - poly caprolactone copolymer except for changing the glycolic acid produced in the same manner, also carrier for cell culture seeding vascular endothelial precursor cells were prepared in the same manner as in example 1. これら各細胞培養担体を実施例1と同様に積み重ねて培養した。 Each of these cell culture support and cultured stacked in the same manner as in Example 1. 本実施例の細胞培養担体は、ポリグリコール酸で構成された多孔質部位がほぼ分解し、消失部分に細胞が増殖されていることが確認された時点、さらにはポリグリコール酸で構成された非多孔質層が分解され始め、各細胞膜の接着が見られた時点においても、前記乳酸−カプロラクトン共重合体で構成された最下位の非多孔質は未分解であった。 Cell culture carrier of this example was composed of polyglycolic acid porous site is almost decomposed, when the cell loss portion is confirmed to be grown, and further composed of polyglycolic acid non porous layer begins to be degraded, even when the adhesion was observed for each cell membranes, the lactic acid - nonporous lowest configured with caprolactone copolymer was undegraded.
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.

本発明の培養担体を利用して、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 carrier 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. また、本発明の培養担体を利用すると、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。 Moreover, the use of culture carrier 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.

培養担体の1構成単位(ユニット)を示した説明図である。 It is an explanatory diagram showing a structural unit (unit) of the culture carrier. 培養担体の2構成単位(ユニット)を積重ね培養担体を示した説明図である。 It is an explanatory view showing a stacking culture carrier 2 structural units (units) of the culture carrier.

符号の説明 DESCRIPTION OF SYMBOLS

1 多孔質層 2 非多孔質層 3 多孔質層 4 非多孔質層 1 porous layer 2 nonporous layer 3 porous layer 4 non-porous layer

Claims (4)

  1. 片面を多孔質層とした生分解性材料の前記多孔質層中に培養により増殖する増殖用細胞を播種した培養担体の複数個を積み重ねて構成された培養用担体。 The porous layer for growth cells configured by stacking a plurality of seeded culture carrier culture carrier that proliferate by culture in biodegradable material in the one side a porous layer.
  2. 培養担体の各培養担体が多孔質層を上面側として積み重ねて構成されたことを特徴とする請求項1記載の培養用担体。 Culture carrier according to claim 1, wherein the respective culture carrier of the culture carrier is configured by stacking the porous layer as the upper surface side.
  3. 請求項1または2に記載の培養用担体を培養液中に浸漬して増殖用細胞の培養を行うことを特徴とする増殖用細胞の培養方法。 The method of culture growth for cells, characterized by immersing the culture carrier according to the culture broth to claim 1 or 2 is cultured in growth for cells.
  4. 培養担体に播種された増殖用細胞が、第1の培養担体では表皮細胞、 前記第1の培養担体に積み重ねられる第2の培養担体では真皮細胞、及び前記第2の培養担体に積み重ねられる第3の培養担体では血管内皮前駆細胞であることを特徴とする請求項3記載の増殖用細胞の培養方法。 Growth for cells seeded in the culture carrier, epidermal cells in the first culture support, the first second dermal cells in culture carrier to be stacked in the culture carrier, and a third which is stacked on the second culture carrier the method of culture growth for cells of claim 3, wherein the in the culture carrier is a vascular endothelial precursor cells.
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JPH07236688A (en) * 1994-02-28 1995-09-12 Terumo Corp Composite material of bioabsorptive plastic and collagen
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JPH07236688A (en) * 1994-02-28 1995-09-12 Terumo Corp Composite material of bioabsorptive plastic and collagen
JP2000507847A (en) * 1996-03-04 2000-06-27 キルシユ,アクセル Coating a molded article and a manufacturing method thereof produced therefrom
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