JP2009247334A - Carrier for cell culture - Google Patents

Carrier for cell culture Download PDF

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JP2009247334A
JP2009247334A JP2008103472A JP2008103472A JP2009247334A JP 2009247334 A JP2009247334 A JP 2009247334A JP 2008103472 A JP2008103472 A JP 2008103472A JP 2008103472 A JP2008103472 A JP 2008103472A JP 2009247334 A JP2009247334 A JP 2009247334A
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cell culture
carrier
cells
cell
culture
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Takao Aoki
Reishi Nishio
玲士 西尾
孝夫 青木
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Toray Ind Inc
東レ株式会社
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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
    • C12M25/00Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/10Hollow fibers or tubes

Abstract

<P>PROBLEM TO BE SOLVED: To solve a problem that, though particle-like beads, hollow fibers and the like are brought into practical use to secure surface areas for adhesion in a large scale culture of adherent cells, those methods are in lack of simplicity with bad efficiency in sowing cells, requirement of specific processes for sowing the cells and the like and are accompanied with difficulty in industrially using the adherent cells. <P>SOLUTION: A carrier for cell culture is provided. The carrier is a solid body made by cutting external portions of a minute hollow body having the external shape of a sphere, an ellipsoid, a polyhedron, a cylinder or a pyramid and a 50-2,000 μm maximum major diameter and a maximum length perpendicular to the maximum major diameter, has an inner wall to a hollow part of which adherent cells can adhere, 5-40% opening on the external shape surface connecting the external space with the hollow part. A method for culturing adherent cells is also provided using the carrier for culturing cells. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

本発明は細胞培養担体および本発明の細胞培養担体を用いて細胞を培養する方法に関するものである。 The present invention relates to a method for culturing cells using the cell culture carrier of the carrier for cell culture and the present invention.

近年、生体からの細胞単離技術、細胞培養技術、細胞保存技術の進歩とともに、細胞を様々な用途に利用する検討が進められている。 Recently, cell isolation techniques from biological, cell culture techniques, together with advances in cell preservation techniques, consider utilizing cells in various applications has been developed. 最も広範に産業利用されている技術は細胞を用いて細胞生育生産物の製造を行うバイオ生産である。 The most widely industrial use has been techniques are bioproduction to manufacture cell growth product with cells. 例を挙げるとインターフェロンの生産や、モノクローナル抗体の生産、各種ウイルスベクターの生産などがあり、これに限らず様々な産業応用がなされている。 By way of example production and of interferon, monoclonal antibody production, include the production of various types of viral vectors, a variety of industrial applications have been made is not limited to this. また、この他にも医薬の開発や生命活動の研究において高価な実験動物に代わりハイスループットで薬物をスクリーニングする方法として細胞を用いたアッセイが行われている。 Moreover, assays using cells has been performed as a method of screening drugs in place high throughput expensive experimental animals in this addition to the study of drug development and life activities also. また、細胞そのものを医療に用いる細胞医療の試みも進められている。 Moreover, trying also underway for cell therapy using cells themselves to a medical.

このように多くの分野で細胞の産業応用が進められているが、これらの試みの多くは浮遊状態で細胞の生存を維持できる非接着系細胞により行われている。 Although Thus industrial applications of cells in many fields has been advanced, many of these attempts have been made by a non-adhesive system cells to maintain cell viability in suspension. これに属さない接着系細胞は細胞の生存を維持するために足場に接着する必要があり、細胞の生存維持、高密度状態での培養・細胞の取り扱いの点において非接着系細胞よりも利便性に劣る。 The adhesive system cells that do not belong to this need to adhere to the scaffold to maintain cell viability, survival of cells, convenience than nonadherent system cells in terms of the handling of the culture, cells in high density conditions inferior to. 例えば、バイオ生産においては可能な限り高密度で細胞を培養することが望ましく、この点で浮遊・懸濁状態での培養が可能な非接着系細胞が好まれる。 For example, it is cultured at high density cells as possible in bioproduction Desirably, nonadherent system cells capable of culture in suspension, suspension at this point is preferred.

近年、ゲノム解析、プロテオーム解析の研究の進展とともに哺乳類細胞や昆虫細胞など多細胞生物の体内より有用な細胞生育生産物が多数発見されているが、これらの細胞生育生産物を製造するためには多細胞生物由来の接着系細胞を必要とする場合もあり、これらの細胞生育生産物の生産には非接着系細胞で用いられている攪拌培養法を直接用いることは出来ない。 Recently, genome analysis, for although useful cell growth product from the body of a mammalian cell or an insect cell such as a multicellular organism with the progress of the study of proteomics has been found a number, to produce these cell growth product is multicellular may require a biological adhesive system cells, it can not be used in the production of these cell growth product directly agitation culture method used in the non-adhesive system cells.

接着系細胞を浮遊状態で生存維持し培養することが出来れば接着系細胞を非接着系細胞と同様に扱うことが可能になるが、このようなことを実現させるために細胞培養担体が考案されている(特許文献1)。 Although it is possible to handle as long as it can survive and maintain culturing adherent system cells in suspension adhesion system cells as well as non-adherent system cells, cell culture carriers have been devised in order to realize such a fact and that (Patent Document 1).

このような細胞培養担体としては、これまでに様々な材質から構成される担体が考案されている。 Such cell culture carrier, a carrier composed of various materials have been proposed so far. 担体上に接着系細胞を接着させ、粒子表面で増殖し、この粒子ごと培養することで、培養槽中の細胞の接着可能な表面積を拡大させ、細胞培養密度を向上させる事ができる。 To adhere the adhesive system cells on the carrier, grown on the particle surface, by culturing this per particle, to expand the bondable surface area of ​​the cells in the culture tank, it is possible to improve the cell culture density.

最初に考案された細胞培養担体は単純な球形の担体であったが、このような担体の表面は凸面の接着面を持ち、そこに接着する細胞は広く伸展した形態をとらざるを得ない。 First is devised cell culture carriers were carriers of a simple spherical surface of such carriers has an adhesive surface of the convex, cells adhere thereto inevitably take widely extended form. このような状態は細胞に正常で無い状態を作り出し細胞の増殖速度、生存率、細胞生育生産物の産生量を低下させることが知られている。 Such conditions are known to reduce the growth rate of the cells create a state not normal to the cell viability, the production of cell growth product. またこのような凸面表面への細胞の初期接着効率は悪く、細胞播種後、細胞を粒子全体に成長させるまでに時間がかかる。 The initial adhesion efficiency of cells to such convex surface is poor, after cell seeding, it takes time to grow the cells in the entire particles.

また、生体内より摘出した哺乳類正常細胞の多くは、細胞の接着、生存、増殖にサイトカインなどの自己産生型可溶性物質を必要とするものも多く、初期に用いられていた細胞培養担体のように細胞播種時に細胞の密度が著しく低くなり、自己産生されたサイトカインが分散してしまう培養方法では細胞の播種、効率的な増殖、培養に困難が伴った。 Also, many mammalian normal cells excised from a living body, cell adhesion, survival, many others require self-producing type soluble substances such as cytokines in proliferation, as carrier for cell culture has been used initially significantly lower density of the cells at the time of cell seeding, the self-produced culture method cytokines resulting in dispersed cell seeding, efficient growth, difficulty has accompanied the culture.

さらに担体表面に細胞を接着させるこれらの細胞培養担体は、懸濁培養する際に、担体と培養槽、または担体同士との接触により細胞が物理的ダメージを受けてしまうことが避けらず、攪拌速度や、培養密度が制限される問題があった。 These cell culture carrier to further adhere the cells to the carrier surface, at the time of suspension culture, regardless avoided that cell by contact with the support and the culture vessel or carrier together, will undergo physical damage, stirring speed and has a problem that the culture density is limited.

細胞培養担体はさらに改良が進められており、細胞の高密度培養を目指してポリウレタンなどを用いた発泡素材により形成された多孔質の細胞培養担体(特許文献2)、セルロース繊維や炭素繊維等(特許文献3)の細胞の入り込む隙間を多数持った小片からなる細胞培養担体などが考案されている。 Cell culture carrier is improved is advanced further, with the aim of high density culture of cells formed by foaming the material with polyurethane porous carrier for cell culture (Patent Document 2), cellulose fibers, carbon fibers or the like ( Patent Document 3) cell cell culture carrier comprising a piece having a large number of gaps which enters the the have been devised.

これらの担体では接着させた細胞の一部が担体の内部の孔に入り込むため、球状の細胞培養担体と比較し、鈍角の接着面による細胞への悪影響や、担体同士や、担体と培養槽との接触による細胞への物理的ダメージを抑えることができる。 Because some cells that are adhered at these carriers from entering the interior of the pores of the support, as compared to the spherical carrier for cell culture, and adverse effects on cells by adhesion surface of the obtuse angle, the support each other and the carrier and the culture vessel it is possible to suppress the physical damage to the cells caused by the contact of.

しかし、この種類の担体は細胞が進入可能な担体内部へと続く孔を厳密に制御することが困難であり、広い内部壁面を持つ担体であっても、その内部壁面へと通じる孔は無秩序に形成されたものであり、培養時には存在する内部壁面のほんの一部のみが利用される。 However, this type of carrier is difficult to strictly control the hole that leads cells to the carrier inside which can enter, even carrier having a wide internal wall, and communicating holes are chaotically to the interior wall surface It has been formed, only a small portion of the inner wall surface present in the culture is used. また、細胞導入経路は無秩序に形成された孔を通じて行うことが必要であり、よく行われる方法としては細胞分散液と担体を混合し、懸濁時に一部が孔に導入される方法が行われる。 Further, cells introduced pathway is required to be done through randomly formed hole, the method is carried out as methods well performed by mixing the cell suspension and the carrier part during suspension is introduced into the hole . これらの効率を向上させるために間欠攪拌法やフィコール法(非特許文献1)が知られるが、これらの方法も満足できる導入効率を持つものでは無く、担体の持つ内部壁面すべてに効率的に細胞を導入できる方法では無かった。 Although intermittent stirring method or a Ficoll method for improving these efficiencies (Non-Patent Document 1) is known, rather those having introduction efficiency even these methods satisfactory, efficient cells to all interior walls with the carrier It was not in a way that can be introduced.

このような細胞導入の困難な細胞培養担体への細胞導入を効率的に行うことを目指し担体内部への溶液の浸透と除去を繰り返し行う方法(特許文献4)や陰圧にしたチャンバーを利用し強制的に細胞を担体内部に導入する方法(特許文献5)などいくつかの方法が考案されているが、導入のための装置が必要であったり、導入のために細胞へのある程度のダメージが予想されるなど、いずれの方法も培養初期段階での作業工程を増加させるものであった。 Such utilizing chamber in which the cell introduction into difficult cell culture support to efficiently repeat method of performing the penetration and removal of the solution to the internal support aims to do (Patent Document 4) and the negative pressure of the cell transfer Although several methods such as a method to force introducing cells therein carrier (Patent Document 5) has been devised, or a need for an apparatus for the introduction, the degree of damage to the cells for introduction etc. is expected, none of the methods were those that increase the work processes in culture early stage.

また、接着系細胞を高密度に培養する方法としては、中空糸を用いた培養がある。 Further, as a method for culturing adherent system cells at a high density, there is a culture using hollow fibers. 束にした中空糸の内部、または外部に細胞を充填し、培養液を循環させることで高密度に集積培養された細胞による細胞生育生産物の生産を行うことができる(特許文献6)。 The hollow yarn was bundles or cells are filled to the outside, the culture solution can be carried out in the production of cell growth product by highly integrated cultured cells by circulating (Patent Document 6). この仕組みを利用し中空糸の中に大量の肝細胞を培養し、患者血液を循環させる「中空糸型人工肝臓」の実現を目指す試みもある。 Culturing a large number of hepatocytes in the hollow fiber utilizing this mechanism, there is also an attempt to aim to realize "the hollow fiber type artificial liver" for circulating the patient's blood.

これらの中空糸を使用した細胞培養方法は高い培養密度を実現可能な方法ではあるが、長い中空糸へ細胞充填は、送液と共に細胞を送りこむことが必要で高度に制御された装置と方法が必要であった。 Although cell culture method using these hollow fibers is a feasible high culture density method, cell-loaded into long hollow fiber, highly controlled apparatus and method requires that feeds the cells with liquid feed is It was necessary. また中空糸内に培養された細胞を培養するためには強制的に細胞培養液を循環させる必要があり、この制約のため、バイオ生産プラント、人工臓器モジュールは、周辺の補助装置も含め、複雑で大規模なものに成らざるを得ない点が問題となっている。 In order to culture the cell culture in a hollow fiber must be forcibly circulate the cell culture, for this limitation, bio-production plants, artificial organs module, including the periphery of the auxiliary device, complex in that it does not inevitably to large ones has become a problem.
特開平4−360682 JP-A-4-360682 特開平4−281784 JP-A-4-281784 特開2004−135668 Patent 2004-135668 特開2006−25635 JP 2006-25635 特開2006−812468 Patent 2006-812468 特開2003−180334 Patent 2003-180334

上記した従来の細胞培養担体には次のような課題がある。 The conventional cell culture carrier as described above has the following problems. すなわち、従来の担体では、担体表面の接着面が凸面であるため、細胞の初期接着が悪く、また、細胞が不自然に伸展した形状をとるため細胞の状況に悪影響がある。 That is, in the conventional carriers, for the adhesive surface of the carrier surface is convex, poor initial adhesion of cells, also have adverse effect on the cellular context for taking cells were unnaturally extended shape. さらに自己産生サイトカインが分散しやすい環境であるため、正常に近い細胞の担体への接着に困難が伴う。 Further, since the self-production cytokine is distributed friendly environment, with difficulty in adhesion to nearly normal cells of the carrier.

また、従来の細胞培養担体では担体の表面に細胞が接着しているため、担体同士、または担体と培養槽が接触した場合の物理的ダメージが避けられない。 Further, in the conventional cell culture carrier for the cells were adhered to the surface of the carrier, it can not be avoided physical damage when the carrier or between carriers and the culture tank are in contact. これらの問題を解決することを目指して発泡素材や繊維素材よりなる改良された細胞培養担体もあるものの、内部の空隙が発泡や繊維状物質を使用して形成された無秩序な構造であるため、空間の利用効率が悪く、また、細胞を高効率で均一に導入することが困難である問題がある。 Although aims to solve these problems some improved cell culture carrier made of foam material or fiber material, because the internal voids are disordered structure which is formed by using a foamed or fibrous material, poor utilization of space, and there is a problem that it is difficult to uniformly introduce the cells at high efficiency.

また、中空糸を用いた接着系細胞の培養方法では高密度の培養が可能であるが、中空糸内への接着系細胞の均一な導入のためには特別の工夫が必要であり、また培養時には強制的な培養液の循環が必要であるため細胞培養部分以外に、送液設備が別に必要となる。 Further, in the method of culturing adhesive systems cells using a hollow fiber is capable of high-density culture, for uniform introduction of the adhesive system cells into hollow fibers within are requires special contrivances, also cultured sometimes in addition to the cell culture portion for circulation is required for forced culture, feeding equipment is separately required.

本発明では上記した課題を解決するため、本発明で示す新規細胞培養担体を考案した。 Because the present invention to solve the problem described above, devised a novel cell culture carrier in this invention.

すなわち、本発明は以下のような構成を有する。 That is, the present invention has the following arrangement.
(1)球体、楕円体、多面体、柱状体または錐体の外部形状を有する、最長径および最長径に垂直な方向の最大長が50〜2000μmの微小中空体の外部を切断した立体であって、中空部分に接着系細胞が接着可能な内壁を有し、外部形状表面に5〜40%の外空間と中空部分をつなぐ開口部を有する、細胞培養担体。 (1) spherical, ellipsoidal, polyhedral, having an external shape of the columnar body or cone, a stereoscopic maximum length in the direction perpendicular to the longest diameter and longest diameter were cut external microballoons of 50~2000μm , has an inner wall adhesion system cells is bondable to the hollow portion has an opening connecting the outer space and the hollow portion of 5-40% to the external shape surface, cell culture carriers.
(2)中空部分がひと続きである(1)に記載の細胞培養担体。 (2) cell culture carrier according to the hollow portion is stretches (1).
(3)1つまたは2つの開口部を有する(1)または(2)に記載の細胞培養担体。 (3) one or with two openings (1) or (2) cell culture carrier according to.
(4)外部形状が柱状体である(1)〜(3)に記載の細胞培養担体。 (4) cell culture carrier according to the external shape is a columnar body (1) to (3).
(5)中空糸を切断することにより製造される請求項1〜4のいずれかに記載の細胞培養担体。 (5) The carrier for cell culture according to claim 1 which is prepared by cutting the hollow fiber.
(6)(1)〜(5)のいずれかに記載の細胞培養担体を用いて接着系細胞を培養する細胞培養方法。 (6) (1) to (5) cell culture method for culturing adhesive system cells using cell culture carrier according to any one of.
(7)細胞培養担体を培地中に平面に並べることを特徴とする(6)に記載の細胞培養方法。 (7) The cell culture method according to, characterized in that arranged in a plane in the medium of the cell culture support (6).
(8)前記中空糸を束ねて切断することで細胞培養担体を培地上に平面に並べることを特徴とする(7)に記載の細胞培養方法。 (8) the method of cell culture according to cut a bundle of hollow fibers, characterized in that arranged in the plane on the medium carrier for cell culture (7).

本発明の細胞培養担体は既存の外周部に細胞を保持する細胞培養担体と比較し、初期の接着効率、細胞の生存率、増殖率、タンパク生産能などにおいて、正常な細胞の状態に近い効果が得られる。 Cell culture carrier of the present invention compared to a cell culture carrier for holding the cells to the existing outer peripheral portion, the initial adhesion efficiency, cell viability, growth rate, such as in protein-producing ability, close to the state of normal cellular effects It is obtained.

本発明の細胞培養担体は、球体、楕円体、多面体、柱状体または錐体の外部形状を有する最長径が50μm〜2000μmの微小中空体の外部を切断した立体であり、中空部分に接着系細胞が接着可能な内壁を有し、外部形状表面の5〜40%の部分に外空間と中空部分をつなぐ開口部を有する定められた形状であれば良い。 Cell culture carrier of the present invention, spherical, ellipsoidal, polyhedral, a solid body longest diameter were cut external microballoons of 50μm~2000μm having an external shape of the columnar body or cone, adhesive system cells to the hollow portion There has bondable inner wall, it may be a shape defined with an opening connecting the outer space and the hollow portion 5 to 40% of the portion of the outer contoured surface.

ここで「形状」とは担体の構成素材が存在する部分の形を示す。 Here shows the shape of the portion there is constituent material of the support is the "shape". 「外部形状」とは担体の「内空間」の立体形状を示す。 The "outer shape" indicates the three-dimensional shape of the "inner space" of the carrier. 内空間とは任意の空間地点(A)において、(A)を通過する任意の3次元内の直線のうち、担体に(A)の両端で交わるものが存在する場合、その地点を担体の「内空間」であると定義する。 In any spatial point and the inner space (A), of the straight line within any three-dimensional passing through (A), in the case where there are those meeting at both ends of the carrier (A), the point of the carrier " defined as the inner space ". また、「内空間」で無い部分を「外空間」と称す。 In addition, the part is not the "inner space" referred to as "outside space".

本発明の細胞培養担体の切断前の外部形状としては、球体、楕円体、多面体、柱状体または錐体であるが、前記外部形状であって、外部応力によって形状が柔軟に変化した形状についても前記外部形状に含まれる。 The external shape before cutting of the carrier for cell culture of the present invention, spherical, ellipsoidal, polyhedral, is a columnar body or cone, it said an external shape, for the shape of the shape is changed flexibly by an external stress the included in the outer shape. ここでいう「柱状体」は円柱、楕円柱または多角柱の形状、「錘体」は円錐、楕円錘または多角錘の形状であると定義する。 Here, the "pillars" are cylindrical, elliptic cylinder or polygonal prism shape, "pyramidal" is defined cone, and the shape of the elliptical weight or polygonal pyramid.

また「内空間」のうち担体では無い部分を「中空部分」と称する。 The free part is the support of the "inner space" is referred to as "hollow portion". 中空部分においては培養液、または細胞が担体外部より進入しうるが再び外空間へと移行する際の自由度が制限されうる空間である。 Broth in the hollow portion, or the cells can enter from the carrier outside a space may be limited flexibility in moving to the outside space again.

本発明の細胞培養担体では、この内空間に中空部分が存在し、担体の形は複雑であるが、外部形状に言及する場合、これらの中空部分により除外される部分については考えない。 The carrier for cell culture of the present invention, the hollow portion is present in this inner space, the shape of the carrier is complicated, when referring to the external shape, not considered for the part to be excluded by these hollow parts. すなわち、担体部分と中空部分をあわせた内部空間を「外部形状」と称す。 That is, the internal space of combined carrier part and the hollow part is referred to as "external shape".

「微小中空体」とは内空間内に担体の構成素材の存在しない部分を有する微小な立体形状を示し、「微小」とはここでは2000μm以下の大きさであることを示す。 It shows the small three-dimensional shape having a non-existent parts of the carrier of the component material into the inner space and "microballoons", indicates that a "fine" here are the following sizes 2000 .mu.m. 構成素材の存在しない中空部分は1つであっても複数であっても良い。 Nonexistent hollow portion of the structure material may be plural be one.

中空部分が複数であるとは、担体内部にお互いに細胞の行き来出来ず連続でない複数の中空空間があることを意味する。 The hollow portion is plural, it means that there is a plurality of hollow spaces not continuous can not traverse cell to each other inside the carrier. 連続部分が存在する場合でも連続部分を通じて細胞が行き来出来ない場合は複数の中空空間が存在するとみなす。 If the continuous portions cells can not traverse through successive portions even if there is deemed a plurality of hollow spaces exist.

「開口部」とは中空部分と外空間の境界面の境界のうち、細胞が通過可能な部位を指す。 Among the boundary of the boundary surface of the hollow portion and an outer space A "opening" refers to a cell can pass sites.

担体内部の空隙率に関する制限はその他の条件を満たす限り無いが、本発明の細胞培養担体を使用する効果である高密度での細胞培養を実現するためには、その空隙率は高い方が好ましく、好ましくは50%以上、最も好ましくは80%以上である。 Restrictions on the porosity of the inner carrier will no only other condition is satisfied, in order to realize the cell culture at a high density is effective to use a carrier for cell culture of the present invention, the porosity is preferably higher , preferably 50% or more, most preferably 80% or more.

ここでの「空隙率」とは「内空間」の中で「中空部分」の占める割合を示す。 The "porosity" herein indicates a ratio of "hollow portion" in the "inner space". 例えば、細胞培養担体の構成素材の占める体積が10であり、中空部分の体積が90、内空間の体積が100である場合、空隙率は90%であると算出される。 For example, a volume 10 occupied by the component material of the carrier for cell culture, the volume of the hollow portion 90, when the volume of the inner space is 100, the porosity is calculated to be 90%.

「内壁」とは中空部分に存在する担体構成素材の表面を示す。 Showing the surface of the carrier structure material present in the hollow portion as "inner wall".

「接着系細胞」とは、細胞の隣接部位に接着面が存在する場合、その接着面に接着する細胞を指す。 By "adhesive system cells", if there is a bonding surface to the adjacent site of a cell refers to a cell to adhere to the adhesive surface. 主に多細胞生物の生体内に見られ、例を挙げると肝細胞、皮膚角質細胞、毛母細胞、口腔上皮細胞、食道上皮細胞、胃粘膜上皮細胞、小腸吸収上皮細胞、大腸吸収上皮細胞、胆管上皮細胞、すい臓インスリン分泌細胞、すい臓グルカゴン分泌細胞、骨細胞、軟骨細胞、平滑筋細胞、心筋細胞、筋肉衛星細胞、神経細胞、ホルモン分泌細胞、白色脂肪細胞、褐色脂肪細胞、骨髄などの初代培養細胞や、ES細胞、iPS細胞または成体幹細胞より分化誘導により作成した接着系細胞が挙げられ、これらに限らないが、壁面との接着形態により細胞活動が大きく影響を受ける細胞に本発明の細胞培養担体を適用することが好適であり、上記した中では肝細胞、口腔上皮細胞、食道上皮細胞、胃粘膜上皮細胞、小腸吸収上皮細胞、大腸吸収上皮細胞 Mainly seen in the body of a multicellular organism, examples and liver cells, skin keratinocytes, the hair matrix cells, oral epithelial cells, esophageal epithelial cells, gastric mucosal epithelial cells, intestinal absorption epithelial cells, colon absorption epithelial cells, biliary epithelial cells, pancreatic insulin secreting cells, pancreatic glucagon secreting cells, bone cells, chondrocytes, smooth muscle cells, cardiac cells, muscle satellite cells, nerve cells, hormone secreting cells, white adipocytes, brown adipose cells, primary, such as bone marrow cultured cells or, ES cells, include adhesive system cells created by differentiation induction from iPS cells or adult stem cells, cells are not limited to, the present invention in cells greatly influenced cell activity by adhesion form the wall it is preferred to apply the culture carrier, hepatocytes in the above-mentioned, oral epithelial cells, esophageal epithelial cells, gastric mucosal epithelial cells, intestinal absorption epithelial cells, colon absorption epithelial cells 胆管上皮細胞、すい臓インスリン分泌細胞、すい臓グルカゴン分泌細胞、ホルモン分泌細胞の初代培養細胞、ES細胞、iPS細胞または成体幹細胞より分化させた接着系細胞が好適である。 Biliary epithelial cells, pancreatic insulin secreting cells, pancreatic glucagon secreting cells, primary culture cells of hormone-secreting cells, ES cells, adhesion system cells differentiated from iPS cells or adult stem cells are preferred. また自然界に存在する細胞にセレクションをかけ作り出した株化細胞、生体内由来の正常細胞や株化細胞に遺伝子導入、遺伝子欠損を生じさせ人工的に作り出した細胞も自然界に存在する細胞と同様に好適に使用できる。 The cell line produced over a selection to cells present in nature, normal cells or cell lines for gene transfer derived from a living body, like the cells present in artificially created cells also naturally cause genetic defects preferably it can be used. 株化接着系細胞の例を挙げると、チャイニーズハムスター卵巣細胞由来のCHO細胞、イヌ腎臓上皮細胞由来のMDCK細胞、マウス胎児皮膚由来のNIH3T3細胞、ラット副腎髄質由来のPC12細胞、ショウジョウバエ由来のS2細胞、蛾由来のSf9細胞、アフリカミドリザル腎臓由来のVero細胞、ヒト子宮ガン由来のHeLa細胞、ヒト結腸ガン由来のCaco−2細胞、ヒト肝ガン由来のHuh7細胞やHepG2細胞がある。 Examples of cell lines adhesion system cells, Chinese hamster ovary cell-derived CHO cells, canine kidney epithelial cell-derived MDCK cells, murine fetal skin-derived NIH3T3 cell, rat adrenal medulla-derived PC12 cells, Drosophila-derived S2 cells there moth-derived Sf9 cells, derived from African green monkey kidney Vero cells, human cervical carcinoma-derived HeLa cells, human colon cancer-derived Caco-2 cells, Huh7 cells, HepG2 cells derived from human liver cancer.

本発明の細胞培養担体は垂直な方向の最大長が50μm〜2000μmであるが、このサイズは接着系細胞を効率よく本発明の細胞培養担体に導入し、かつ、空間を効率的に使用するために適した大きさである。 Since the carrier for cell culture of the present invention is the maximum length in the direction perpendicular is a 50Myuemu~2000myuemu, this size is to introduce the adhesive system cells to the cell culture support efficiently the present invention, and, for efficient use of space the size is suitable for. すなわち50μm以下のサイズであっては、個々の担体への細胞導入数が限られ培養時の空間の利用効率が低下する。 That a size below 50μm, the use efficiency of space during cell number introduction into individual carriers is limited culture decreases. また、培養時に培地との分離が困難となり効率が低下する。 Also, separation of the medium efficiency becomes difficult to decrease the time of culture. また2000μm以上のサイズを持つものは均一な細胞の導入に困難が伴い、内部の形状にもよるが空間の利用効率が低下する。 Also those having a size of more than 2000μm involves difficult the introduction of homogeneous cell, depending on the internal shape decreases the utilization efficiency of space. 好ましくはサイズが50μm〜1000μmの小片であり、もっとも好ましくは100μm〜500μmである。 Preferably the size is small piece of 50Myuemu~1000myuemu, most preferably 100Myuemu~500myuemu.

本発明の細胞培養担体は開口部が5〜40%であるが、この性状は担体の中空部分と外空間との培養液の循環を部分的に抑制し、自己産生サイトカイン濃度を向上させること、また、担体への細胞導入時に必要な構成である。 Although the carrier for cell culture of the present invention is an opening 5 to 40%, and this property is that the circulation of the culture liquid of the hollow portion and an outer space of the carrier is partially suppressed, improving the self-producing cytokine concentration, Further, it is necessary configuration upon cell transfer to the carrier. 細胞培養担体の開口部が5%未満である場合、開口部が小さすぎるため細胞播種時の導入効率が低下し、また、細胞培養担体の開口部が40%を上回る場合も、細胞播種時の効率が低下し、また自己産生サイトカインの濃度上昇も期待できないため、好ましくない。 If the opening of the cell culture carrier is less than 5%, the introduction efficiency during cell seeding reduced because the opening is too small, also when the openings of the carrier for cell culture is greater than 40%, of the time of cell seeding efficiency is lowered, since the increase in concentration of the self-producing cytokines can not be expected, which is not preferable.

本発明の細胞培養担体の外部形状は様々な形状が考えられるが、球形の外部形状を有する微小中空体を考えた場合、球形中空体上部に球状の開口部が存在する場合、球の半径の半分の半径を有する開口部が存在すると、開口部の表面積の割合はおよそ6.6%となり、これより遙かに小さい開口部を持つ細胞培養担体は細胞導入時の効率が著しく低下してしまう場合がある。 The outer shape of the carrier for cell culture of the present invention are conceivable various shapes but, when considering the hollow microspheres having a spherical outer shape, if there is the opening of the spherical spherical hollow body top, the sphere radius When opening having a radius of half present, the ratio of the surface area of ​​the openings becomes about 6.6%, than this much small cell culture support having an opening portion decreases remarkably efficiency at cell transfer If there is a. また球状の中空体を中央部にて平面で切り取った半球の細胞培養担体を考えた場合、担体の厚みを考えなければ、開口部はおよそ33%となり、この形状よりも開口部が広い場合は形状によっては細胞の播種時の効率は高く保てるものの、細胞培養時の自己産生サイトカインの局所濃度を上昇する効果が得られにくい場合がある。 Also when considering the carrier for cell culture of the hemisphere taken in a plane a hollow body of spherical at the center portion, to be considered the thickness of the carrier, the opening becomes about 33%, when the opening portion than the shape is wide although depending on the shape kept high efficiency during cell seeding, the effect is difficult to obtain for increasing the local concentration of the self-producing cytokines during cell culture. したがって、本発明の細胞培養担体では開口部が外部形状面積に対し5〜40%であることが必要であるが、細胞培養担体の形状を考慮した場合、好ましくは5〜35%、より好ましくは10〜30%である。 Thus, although the carrier for cell culture of the present invention it is necessary that the opening is 5 to 40% with respect to the outer shape area, when considering the shape of the cell culture carrier, preferably 5 to 35%, more preferably 10 to 30%.

本発明の細胞培養担体の構成する構成素材に関しては、本発明の細胞培養担体の形状に成型可能なあらゆる素材が使用可能であり、素材の調整方法、平均分子量等制限はないが、好ましくは金属または高分子物質を利用することができる。 For the constituent material constituting the carrier for cell culture of the present invention are available moldable any material in the shape of a cell culture carrier of the present invention, a method of adjusting the material, but such is not limited average molecular weight, preferably a metal or utilize a polymeric material. ここで高分子とはゴム、多糖類、タンパク質、水溶性合成高分子、有機溶媒可溶性高分子が挙げられ、具体例を挙げるとセルロース、アラビアガム、トラガガントガム、カラギーナン、アガー、グアガム、カラヤガム、ローカストビーンガム、ペクチン、ガラクタン、マンナン、プルラン、キサンタンガム、ゼラチン、カゼイン、コンドロイチン硫酸、コラーゲン、エラスチン、フィブロイン、キトサン、キチン、ヒアルロン、カルボキシメチルデンプン、メチルヒドロキシプロピルデンプン、アルギン酸エステル、アルギン酸塩、ポリビニルアルコール、ポリビニルピロリドン、ポリビニルエチルエーテル、カルボキシビニルポリマー、ポリアクリル酸、ポリアクリルアミド、ポリスチレン、ポリ塩化ビニル、ポリ酢酸ビニル、ポリアクリ Here rubber and polymer, polysaccharides, proteins, water-soluble synthetic polymer, and organic solvent-soluble polymer, specific examples and cellulose, gum arabic, Toragagantogamu, carrageenan, agar, guar gum, karaya gum, locust bean gums, pectins, galactans, mannans, pullulan, xanthan gum, gelatin, casein, chondroitin sulfate, collagen, elastin, fibroin, chitosan, chitin, hyaluronic, carboxymethyl starch, methylhydroxypropyl starch, alginic acid esters, alginates, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl ethyl ether, carboxyvinyl polymers, polyacrylic acid, polyacrylamide, polystyrene, polyvinyl chloride, polyvinyl acetate, polyacrylic ニトリル、ポリスルホン、ポリウレタン、メタクリル酸アクリル、フッ素系高分子、ポリアミド、ポリピペラジンアミド、ポリウレア、架橋ポリエーテル、ポリアクリロニトリルなどがある。 Nitrile, polysulfone, polyurethane, acrylic methacrylic acid, fluorine-based polymers, polyamides, polypiperazine amides, polyureas, crosslinked polyether, and the like polyacrylonitrile. 好ましくはポリスルホン、メタクリル酸アクリルであり、より好ましくは本発明の実施例で使用されているポリスルホンである。 Preferably polysulfone, a methacrylic acid acrylic, more preferably polysulfone used in embodiments of the present invention.

さらに本発明の細胞培養担体は単一の構成素材から構成されても良いし、細胞接着面だけ別の構成素材より構成されるなど複数の素材から構成されていても良い。 It further carrier for cell culture of the present invention may be composed of a single component material may be composed of a plurality of materials such as composed of different constituent material just cell adhesion surface. 例えば接着系細胞の接着性を向上させるために担体全体の表面または担体内部の表面のみを細胞接着性を向上させる別の構成素材でコーティングすることは好ましい。 For example by coating only the surface of the inner surface or support the whole carrier in order to improve the adhesion of the adhesive system cells by another constituent material for improving the cell adhesion preferred. コーティング素材としては特に制限されるものでは無いが、コラーゲン、ゼラチン、フィブロネクチンによるコーティング、正電荷、負電荷の官能基の導入、各種の細胞接着因子、例えば、RGDペプチド(Arg−Gly−Asp)による修飾などは好ましい。 There is no particular limitation on the coating material, collagen, gelatin, coating with fibronectin, positive charge, introduction of the functional groups of negative charge, various cell adhesion factors, for example, by RGD peptide (Arg-Gly-Asp) modification such as preferred.

また本発明の細胞培養担体は、それぞれに開口部を持つ複数の内空隙が存在しても良いが、内空間において中空部分がひと続きである場合、すなわち中空部分が1つのみ存在する場合に細胞の導入効率、内部空間の効率的な利用が実現できるため高い性能を発揮することができるため、好ましい。 The carrier for cell culture of the present invention, a plurality of inner voids having openings each may be present, if the hollow portion in the inner space is a stretch, that is, if the hollow portion there is only one transduction efficiency of a cell, it is possible to achieve high performance for efficient use of the internal space can be achieved, preferred. ただし本発明の細胞培養担体のような微細な担体を多数個生産する過程では、製造時に生ずる個体差により一部の担体がひと続きでは無い内空間を有する場合もあり、このような場合も細胞培養担体の平均の中空部分の個数が1.5以下である場合はすべての細胞培養担体がひと続きの中空部分を持つ場合と同様の効果が得られるため好ましく、より好ましくは平均の中空部分の個数が1.1以下である。 However, in the course of a large number producing fine carriers such as carrier for cell culture of the present invention is also a part of the carrier by an individual difference created during manufacture with a space among not a stretch, the cells also in such a case preferably for the same effect as in the case when the number of the hollow portion is 1.5 or less where all of the cell culture support having a hollow portion of the stretch is obtained average of culture carrier, more preferably an average of the hollow portion the number is 1.1 or less.

また本発明の細胞培養担体は、中空部分と外空間をつなぐ開口部が1つまたは2つであることが好ましい。 The carrier for cell culture of the present invention is preferably an opening portion connecting the hollow portion and an outer space of one or two. 開口部の個数は細胞培養担体の中空部分を通過する細胞、および細胞培養液の移動に関わっており、開口部が一つまたは2つである場合は細胞導入時の効率を高くするために有効であり、また担体内部の自己産生サイトカインの濃度を高く保つことができる。 Cells The number of openings passing through the hollow portion of the cell culture support, and are involved in the movement of the cell culture, if the opening is one single or 2 effective to increase the efficiency during cell transfer , and the addition can be kept high concentration of self-producing cytokines internal support. また開口部が2つの場合であっても細胞導入時に片側の開口部を閉鎖した状態を作り、高い効率で細胞を導入することが可能である。 Further, even if the opening is two create a state of closing the one side of the opening at the time of cell transfer, it is possible to introduce the cells at high efficiency. ただし多数個製造した担体のうち、製造時のばらつきにより一部が2以上の開口部を有する場合もその全体の平均数が2.5以下である場合は同様の機能が得られるため好ましく、より好ましくは平均が2.2以下である場合である。 However among many pieces produced carrier, preferably for the case part is the average number of the entire case having two or more openings than 2.5 obtained similar functionality due to variations in manufacturing, and more preferably when the average is 2.2 or less. なお、中空部分と外空間をつなぐ部分のうち、構成物が存在しないが、細胞が透過できるほどの大きさの無い微細な孔構造の存在は制限されるものではない。 Of the portion connecting the hollow portion and an outer space, but there is no arrangement, the presence of about the size of no fine pore structure cells can pass is not intended to be limited. 細胞の種類や培養方法によっては、これらの細胞が透過出来ない孔は培養液の流通を促進するため望ましいことがある。 Depending on the type and method for culturing cells, holes these cells can not be transmitted may be desirable to facilitate the circulation of the culture solution.

また本発明の細胞培養担体は、形状が柱状体であることが好ましい。 The carrier for cell culture of the present invention, it is preferable shape is columnar body. 本発明の細胞培養担体に効率的に細胞を播種するためには後述するように密集した状態で開口部を揃えた状態をとらせることが好ましい使用方法であるが、柱状体はこの用途において適した形状であり、より好ましくは図2に示す柱状体の外形を有する細胞培養担体である。 Although to seed efficiently cells to the cell culture support of the present invention is a method preferably used to assume a state in which aligned openings in a state of dense as described below, the columnar body suitable in this application and the shape, more preferably a cell culture support having a contour of the columnar body shown in FIG. ただし端部には微細な例外部分が存在することは本体部分が柱状体であるかぎり許容される。 However it is to the end there is a fine exception moiety is acceptable as long as the body portion is columnar body. 柱状体の直径としては100〜1000μmであることが好ましく、200〜500μmであることがより好ましい。 Preferably the diameter of the columnar body is 100 to 1000 [mu] m, and more preferably 200 to 500 [mu] m.

本発明の細胞培養担体の製造方法としては、鋳型を用いた製造や、リソグラフィーによる製造、類似構造を持った自然界天然構造物の加工、前駆構造物の切断による製造が挙げられる。 As a method for producing a cell culture carrier of the present invention, manufacturing and using a mold, production by lithography, processing of natural natural structure having a similar structure include preparation by cleavage of the precursor structure. 中でも、本発明の細胞培養担体の好ましい態様である、前記柱状体の形状を有する細胞培養担体は、中空糸を切断することによって好ましく製造される。 Among them, a preferred embodiment of the carrier for cell culture of the present invention, the cell culture support having a shape of the columnar body is preferably produced by cutting the hollow fiber. 例えば、長さ100000μmの形状を有する中空糸を切断した場合、長さ50μmの本発明の細胞培養担体を2000個製造することができる。 For example, when cutting the hollow fiber having the shape of length 100000, it can be 2,000 producing the carrier for cell culture of the present invention the length 50 [mu] m. 製造効率を考えた場合、中空糸の長さは長い方が好ましく、好ましくは最終的に得られる細胞培養担体の最長径に垂直な方向の最大長の100倍以上の長さの中空糸を切断して生産することであり、より好ましくは1000倍以上の長さの中空糸を切断して生産する方法である。 When considering manufacturing efficiency, the length of the hollow fibers longer is preferred, preferably ultimately longest diameter of the obtained cell culture support to cut the hollow fibers perpendicular direction of maximum length 100 times or more in length and it is to produced, and more preferably a method of producing by cutting the hollow fiber length of more than 1000 times.

また中空糸から細胞培養担体を作製する段階で、中空糸を束ねた状態で切断することも好適な製造方法の1つである、開口部の方向を揃えた細胞培養担体が密集した集合体を効率的に生産することができる。 Also at the stage of producing a cell culture carrier from the hollow fiber, it is also one of preferable production methods of cutting in a state in which a bundle of hollow fibers, the assembly cell culture support having uniform direction of the opening portion is densely it can be produced efficiently. 中空糸を束ねて切断することにより作製した後は、そのまま細胞培養担体として用いることもできるし、表面修飾、片方の開口部を閉じるなどの処理を行い細胞培養担体として用いることもできる。 After preparing by cutting a bundle of hollow fibers, it can either be used as a carrier for cell culture, can be used as a surface-modified, cell culture carrier performs processing such as closing the opening of one.

また本発明は前記細胞培養担体を用いて接着系細胞を培養する細胞培養方法に関する。 The present invention also relates to a cell culture method for culturing adherent system cells using the cell culture support. ここでいう細胞培養とは、酸素、栄養素等を含む培養液等を介して担体内部に保持した接着系細胞の生存を維持することを示す。 The cell culture here show that maintaining oxygen, the survival of the adhesive system cells held within the carrier through the culture medium or the like containing nutrient. また、培養の形態としては、細胞培養に一般的に用いられる培養槽を使用することができ、具体的にはペトリ皿、プラスチックプレート、プラスチックチューブ、ガラスチューブ、中空糸繊維、スパイラルフィルム、カラム、マルチウェルプレート、マルチシートプレート、細胞生育生産物生産のために用いられる攪拌培養用の培養チャンバー、各種細胞アッセイ機材の細胞保持部分などが挙げられるがこれらに限らず、細胞の生存に適した環境であれば良い。 As the form of the culture, a culture vessel generally used can be used for cell culture, specifically a petri dish, a plastic plate, a plastic tube, glass tube, hollow fiber fiber, spiral film, column, multi-well plates, multi-sheet plate, the culture chamber for stirring culture used for cell growth product production, such as a cell holding part of the various cell assay equipment and the like is not limited to, an environment suitable for cell survival it is sufficient. また、細胞培養に使用する培地についても、細胞の生存に適した培地であれば特に制限されない。 As for the medium used for cell culture is not particularly limited as long as it is a medium suitable for cell survival.

本発明の細胞培養方法においては、前記細胞培養担体の形状的特徴を活かすことで、前記細胞培養担体を培地中に平面に並べることができる。 In the cell culture method of the present invention, the by leveraging the geometrical characteristics of the carrier for cell culture, can be arranged in the plane of the cell culture carrier in the medium. ここでいう「培地中に平面に並べる」とは、細胞培養担体の大部分が重ならずに整列していることであり、例えば、円筒状の培養槽の外壁に整列した状態も含まれる。 The "arranged in a plane in the medium" is to aligned without overlapping the majority of the cell culture support, for example, also include a state of being aligned with the outer wall of the cylindrical culture vessel. 柱状体の細胞培養担体を密集した状態で開口部の方向を揃えた状態にして細胞を播種した場合、接着系細胞を播種する過程で高い効率をもって細胞を細胞培養担体に接着させることが可能であり本発明の細胞培養担体を効率的に利用することができるため好ましい。 While confluent cell culture carrier of the columnar body in the state of aligning the direction of the opening when seeded with cells with high efficiency in the process of seeding adhesive system cells cells can be adhered to the carrier for cell culture It preferred because there can be utilized the carrier for cell culture of the present invention efficiently. 細胞培養担体を、開口部を揃えて密集した状態で集合させる方法としては、開口部を揃え密集した状態の集合として製造する方法や、作製した細胞培養担体を後から集合させる方法が考えられるが、好ましくは密集した集合として作製する方法である。 The carrier for cell culture, as a method to aggregate in a state of dense aligned openings, and a method of manufacturing a set of dense state aligned openings, a method to aggregate later cell culture carrier produced is considered is a method preferably prepared as set dense.

本発明の細胞培養担体に細胞を導入する方法に関しては特に限定されず、細胞培養担体の開口部に細胞懸濁液を滴下して重力により導入する方法、細胞培養担体に細胞懸濁液を加えて遠心力を用いて導入する方法が挙げられるが、細胞培養担体を培地中に平面に並べた場合、開口部を固定面と反対方向の一方向に向けて整列させておくことで、上部より細胞懸濁液を滴下することで、重力により細胞が効率よく細胞培養担体に導入されうる。 Is not specifically defined, the method of introducing the cells into the cell culture carrier of the present invention, a method of cell suspension into the opening of the cell culture carrier dropwise to the introduction by gravity, the cell suspension to the cell culture carrier added Te may be mentioned a method of introducing using a centrifugal force, when arranged in a planar carrier for cell culture in the medium, by leaving aligned towards the opening in one direction of the fixed surface opposite direction from upper portion by dropping the cell suspension, the cells can be efficiently introduced into the cell culture support by gravity.

本発明の細胞培養方法の好ましい態様である、細胞培養担体を培地中に平面に並べる方法に関しては特に限定されず、培養槽平面に一時的に固定する方法、磁気や重力、遠心力等などを用いて培養槽に固定せずに整列させる方法が挙げられるが、好ましくは培養槽平面に一時的に固定する方法である。 Is a preferred embodiment of the cell culture method of the present invention is not specifically defined, the method of arranging the planar carrier for cell culture in a medium, a method of temporarily fixing the culture tank plane, magnetic and gravity, and centrifugal force, etc. the method for aligning without fixing to the culture vessel and the like used, but a method preferably temporarily secured to the culture tank plane.

なお本発明の細胞培養方法においては、細胞培養のために本発明の細胞培養担体に接着させた接着系細胞を凍結保存する場合も細胞培養の一部である。 In still the cell culture method of the present invention, which is part of a cell culture may cryopreserving adhesive system cells adhered to the cell culture support of the present invention for cell culture. 凍結の方法は問わないが、好ましくは−80℃以下での凍結であり、より好ましくは−150℃以下での凍結である。 Although not limited freezing method is preferably frozen at -80 ° C. or less, more preferably freezing at -150 ° C. or less.

以下に本発明の詳細を説明するため実施例を挙げるが、本発明は実施例に限定されるものではない。 Examples are given for explaining details of the present invention are shown below, but the present invention is not limited to the examples.

実施例1 細胞培養担体の作成 <方法> Creating Example 1 cell culture carrier <method>
外径400μm、内径300μmのポリスルホン中空糸(東レ、“トレビーノ”用)を隙間の空かないように束ねた後、凍結切片封入剤(ティッシュテックOCTコンパウンド(サクラファインテック社))に浸沈させ、十分にコンパウンドが浸透するまで数時間静置した。 Outer diameter 400 [mu] m, after bundled so as not empty polysulfone hollow fibers having an inner diameter of 300μm (the Toray, for "Torebino") clearance, is Hita沈 frozen sections encapsulant (Tissue Tech OCT compound (Sakura Finetech, Ltd.)), enough compound was allowed to stand for several hours to penetrate. 束ねた状態を保ったまま−150℃にて凍結し、十分に凍結させた凍結ブロックを凍結切片作成機(OTF5000(ブライト社英国))にて−20℃で、カーボンブレード(ミクロトーム用替え刃C35(フェザー社))をもちいて20μmの厚さに切断した。 Frozen at -150 ° C. while maintaining the bundled state, at -20 ° C. at sufficiently frozen were frozen block Frozen sections making machine (OTF5000 (Bright Co. UK)), carbon blade (microtome for replacement blades C35 It was cut to a thickness of 20μm using a (Feather Co., Ltd.)). 切断した中空糸集合体は、水溶性接着剤(シリル化ウレタン樹脂接着剤(“ボンドニューハイテック”(コニシ)))を薄く塗布したスライドガラス(シランコーティングスライドS3003(Dako))上に固定し、乾燥させた。 Cut hollow fiber assemblies, fixed on a water-soluble adhesive (silylated urethane resin adhesive ( "Bond New Hitec" (Konishi))) thinly coated glass slides (silane coated slides S3003 (Dako)), and dried.

スライドガラス上で十分に乾燥させた細胞培養担体は純水に数時間浸して切断時に使用した凍結切片封入剤を除去した。 Carrier for cell culture was sufficiently dried on a glass slide was removed cryosections encapsulant used during cutting to soak for several hours in pure water. その後、細胞培養担体は固定したスライドガラスと共にオートクレーブにて120℃、20分間処理を行い滅菌し、使用まで室温で保存した。 Thereafter, 120 ° C. cell culture carrier in an autoclave with a fixed glass slides, were sterilized for 20 minutes and stored at room temperature until use.

<結果> <Result>
作成した細胞培養担体の電子顕微鏡写真(日立S4800)を図3Aに、実体顕微鏡により撮影した写真を図3Bに示す。 Electron micrographs made by cell culture carrier (Hitachi S4800) in FIG. 3A, shows a photograph taken by a stereoscopic microscope in Figure 3B. この細胞培養担体は外径が400μm、内径が300μm、高さがおよそ300μmであり開口部の比率はおよそ29%である。 The carrier for cell culture outer diameter of 400 [mu] m, an inner diameter of 300 [mu] m, is approximately 300 [mu] m height ratio of the openings is approximately 29%. 細胞培養担体は開口部を上下に向けた状態で密な状態でスライドガラス上に固定されており(図3C)、切断やオートクレーブ処理による乱れは生じなかった。 Cell culture carrier is fixed on a slide glass dense state while with its opening vertically (FIG. 3C), disturbance due cutting or autoclaving did not occur. また、スライドガラス上に水溶性接着剤により固定した細胞培養担体は数時間、細胞培養培地に浸した状態を経た後は、弱い水流などにより容易にスライドガラス上から遊離し、分散状態になった(図3D) Also, several hours fixed cell culture support by water-soluble adhesive on the slide glass, after a state immersed in the cell culture medium is easily released from the glass slide or the like weak water flow became dispersed state (Fig. 3D)
実施例2 細胞培養担体へのCaco―2細胞の播種 10cm細胞培養プレート(ファルコン社)上にて培養培地(DMEM/10%FBS(ギブコ社))にて培養したCaco−2細胞(ヒト結腸由来株化細胞細胞)を細胞剥離溶液(0.25%トリプシン/EDTA(シグマ社))処理しプレートから遊離させた。 EXAMPLE 2 Caco-2 cells seeded 10cm cell culture plates (Falcon) culture medium at the top (DMEM / 10% FBS (Gibco)) Caco-2 cells cultured in (derived from human colon into the cell culture carrier cell line cells) cell detachment solution (0.25% trypsin / EDTA (sigma)) to liberate from the treated plate. 培養培地にて2回洗浄し残存する細胞剥離溶液を除去した。 To remove cell detachment solution remaining was washed twice with culture medium. 1000万個のヒト結腸ガン由来Caco−2細胞を10mlの培養培地に懸濁した。 Ten million human colon cancer-derived Caco-2 cells were suspended in culture medium 10 ml.

細胞培養担体を密に整列させたスライドガラスは10cm細胞培養プレート(ファルコン社)中央に配置し、0.3%細胞培養用I型コラーゲン溶液(“セルゲン”(高研))を滴下し、常温で2時間静置し、細胞培養担体表面をコラーゲンでコートした。 Slides were closely align the cell culture carrier is disposed in 10cm cell culture plates (Falcon) center, was added dropwise 0.3% cell culture type I collagen solution ( "Serugen" (Koken)), room temperature in standing 2 hours, the cell culture carrier surface was coated with collagen. 添加したコラーゲン溶液を除去し、滅菌したリン酸緩衝液で細胞培養担体を洗浄した。 Collagen solution was removed was added to wash the cell culture carrier in sterile phosphate buffer. 細胞培養担体の上部より少量ずつCaco−2細胞分散液を滴下した。 Portionwise from the top of the cell culture support was added dropwise Caco-2 cell suspension. 添加後、30分間静置しCaco−2細胞が底に沈み、細胞培養担体の中に導入されるのを待ち、細胞培養担体を固定したスライドガラスを50mlチューブ(コーニング社)に移し、培養培地を満たした状態で、スライドガラスが垂直になる状態を保って培養インキュベーター(NAPCO CO26000)の中で37℃、CO 濃度5%を保ち24時間培養した。 After the addition, it sinks stand to Caco-2 cells bottom 30 minutes, waiting to be introduced into a cell culture carrier, transferred to glass slides and fixed cell culture carrier 50ml tubes (Corning), the culture medium in a state filled, 37 ° C. in a culture incubator while maintaining the state in which the slide glass is perpendicular (NAPCO CO26000), were cultured for 24 hours maintaining the CO 2 concentration of 5%.

24時間後、50mlチューブのふたを開け、穏やかに溶液を懸濁することでスライドガラスより細胞培養担体を遊離させた。 After 24 hours, open the lid of 50ml tubes were gently solution to release the cell culture support from the glass slides by suspending. 細胞培養担体を除去したスライドガラスは取り除き、50mlチューブ内を3000回転/分、5分間遠心し細胞培養担体を沈殿させ、培養液を取り除く作業を3回行った。 Slides removing the cell culture carrier is removed and 3000 rev / min in 50ml tube, centrifuged to precipitate the cell culture carrier 5 minutes was performed 3 times the task of removing the culture medium.

<結果> <Result>
Caco−2細胞を接着させた細胞培養担体の凍結切片を作成した図4Bに、細胞を接着させた細胞培養担体の走査型電子顕微鏡にて観察した像を図4Aに示す。 Figure 4B that created the frozen sections of the cell culture carrier to adhere the Caco-2 cells, indicating the image was observed by a scanning electron microscope of the cells were allowed to adhere carrier for cell culture in FIG. 4A.
細胞培養担体の上部よりCaco−2細胞懸濁液を滴下することにより、一部の細胞が細胞培養担体の内部に導入された。 By dropping the Caco-2 cell suspension from the top of the cell culture carrier, a portion of the cells were introduced into the cell culture support. その後、細胞培養担体の内壁に接着させるために細胞培養担体を固定したスライドガラスを垂直に保ち、培養することで、内部に導入されたCaco−2細胞が細胞培養担体内部に生着することが確認された。 Then, keeping the slide glass were fixed cell culture carrier for adhering to the inner wall of the cell culture support vertically, by culturing, be Caco-2 cells introduced therein engrafted inside the cell culture support confirmed.

実施例3 細胞培養担体を用いた接着系細胞の懸濁培養 <方法> Suspension cultures Bonded Cells Using Example 3 cell culture carrier <method>
Caco−2細胞を内壁に接着させた細胞培養担体とCaco−2細胞単独の攪拌浮遊培養時の細胞生存率の推移を比較評価した。 Caco-2 cells were comparatively evaluated changes in cell viability at the time of stirring suspension culture carrier for cell culture and Caco-2 cells alone adhered to the inner wall.

実施例2で調製した細胞培養担体と、実施例1と同様の方法で10cm細胞培養プレートで培養し、細胞剥離溶液にて回収したCaco−2細胞を、それぞれ180G、5分間の遠心を3回行い、細胞を洗浄した後、それぞれの細胞を6つのチューブにわけ、培養インキュベーターの中に設置した転倒混和型ローテーターにより30回転/分の速度で懸濁培養を行った。 A carrier for cell culture prepared in Example 2, were cultured in 10cm cell culture plates in the same manner as in Example 1, the Caco-2 cells were recovered by cell detachment solution, 3 times with centrifugation at each 180G, 5 minutes performed after the cells were washed, each of the cells divided into six tubes, the suspension culture at 30 rev / min was carried out by mixing by inversion type rotator was placed in the culture incubator. そして培養開始後0、1、3、6、24、48時間後の細胞を回収し180G5分間の遠心で細胞および細胞培養担体を沈殿させた後、培養液を除去し−30℃で凍結保存した。 And after precipitating the cells and cell culture carrier cells 0,1,3,6,24,48 hours after initiation of the culture by centrifugation of the collected 180G5 minutes and stored frozen at removing -30 ° C. The culture . すべてのサンプルがそろった48時間後、サンプルを解凍し細胞生存率測定キット(セルカウンティングキット(WST−8)(同仁化学))とプレート吸光度計(マルチスキャンJX(サーモラボラトリー社))を用いて細胞の生存量を測定した。 48 hours after all of the samples are aligned, samples were thawed cell viability assay kit (Cell Counting Kit (WST-8) (Dojin Chemical)) using a plate absorption meter (Multiscan JX (Thermo Laboratory)) It was measured survival of cells.

<結果> <Result>
それぞれの細胞生存率の変化を図5に示す。 The changes in each cell viability is shown in Figure 5. 細胞培養担体を用いないCaco−2細胞の生存率はアッセイ開始直後より急激に減少し24時間後には20%以下となった。 Viability of Caco-2 cells without using a carrier for cell culture to more rapidly reduced 24 hours after the immediately assay initiation was 20% or less. これに対し、細胞培養担体に接着させたCaco−2細胞の生存率は培養開始後にある程度の減少が見られるが、その後、上昇傾向にあり、細胞培養担体内での生存が維持され細胞増殖が起きていた。 In contrast, although the survival rate of the Caco-2 cells were allowed to adhere to the cell culture support is seen to some extent reduced after initiation of the culture, then on the rise, survival in a cell culture carrier is maintained cell proliferation It had occurred.

本発明の細胞培養担体の要件を満たす形状の例。 Examples of shapes that meet the requirements of the cell culture carrier of the present invention. AとBの2種類の例を示す。 It shows two examples of A and B. 上図の三次元概念図において斜線部分は内壁面を示す。 Hatched portion in the three-dimensional conceptual diagram of the figure shows the inner wall surface. 下図は上図の断面概念図、細胞 Conceptual cross-sectional view of the figure below the figure, cells 本発明の細胞培養担体の要件を満たす形状の例、A、B、C、D、E、Fの6種類を示す。 Examples of shapes that meet the requirements of the cell culture carrier of the present invention, A, B, C, D, E, and six F shown. 図の三次元概念図において斜線部は内壁面を示す。 Hatched portion in the three-dimensional conceptual diagram of the figure shows the inner wall surface. 実施例1により作成した細胞培養担体の走査型電子顕微鏡写真(A)、実体顕微鏡写真(B)、スライドガラス上に整列させた細胞培養担体(C)、水溶液に分散させた細胞培養担体(D)である。 Scanning electron micrographs of carrier for cell culture were prepared by Example 1 (A), the entity micrograph (B), the aligned carrier for cell culture on a glass slide (C), a cell culture carrier which is dispersed in an aqueous solution (D ) it is. 実施例2によりCaco−2細胞を接着させた細胞培養担体の走査型電子顕微鏡写真(A)、凍結切片の可視光観察像(B)である。 Scanning electron micrograph of the Example 2 was adhered to Caco-2 cell cell culture carrier (A), a visible light observation image of frozen sections (B). 実施例3による本発明の細胞培養担体と、細胞単独の攪拌培養時のCaco−2細胞生存率の変化結果である。 A carrier for cell culture of the present invention according to Example 3, a Caco-2 changes the results of cell viability when cells alone spinner culture.

Claims (8)

  1. 球体、楕円体、多面体、柱状体または錐体の外部形状を有する、最長径および最長径に垂直な方向の最大長が50〜2000μmの微小中空体の外部を切断した立体であって、中空部分に接着系細胞が接着可能な内壁を有し、外部形状表面に5〜40%の外空間と中空部分をつなぐ開口部を有する、細胞培養担体。 Spherical, ellipsoidal, polyhedral, having an external shape of the columnar body or cone, a stereoscopic maximum length in the direction perpendicular to the longest diameter and longest diameter were cut external microballoons of 50 to 2000 m, the hollow portion adhesive system cells having a bondable inner wall has an opening connecting the outer space and the hollow portion of 5-40% to the external shape surface, the cell culture carrier.
  2. 中空部分がひと続きである請求項1に記載の細胞培養担体。 The carrier for cell culture according to claim 1 hollow part is stretch.
  3. 1つまたは2つの開口部を有する請求項1または2に記載の細胞培養担体。 One or two cell culture carrier according to claim 1 or 2 having an opening.
  4. 外部形状が柱状体である請求項1〜3のいずれかに記載の細胞培養担体。 The carrier for cell culture according to claim 1, wherein the external shape is a columnar body.
  5. 中空糸を切断することにより製造される請求項1〜4のいずれかに記載の細胞培養担体。 The carrier for cell culture according to claim 1 which is prepared by cutting the hollow fiber.
  6. 請求項1〜5のいずれかに記載の細胞培養担体を用いて接着系細胞を培養する細胞培養方法。 Methods Cell culture for culturing adhesive system cells using cell culture carrier according to any one of claims 1 to 5.
  7. 細胞培養担体を培地中に平面に並べることを特徴とする請求項6に記載の細胞培養方法。 The cell culture method according to claim 6, characterized in that arranged in a plane in the medium of the cell culture carrier.
  8. 前記中空糸を束ねて切断することで細胞培養担体を培地上に平面に並べることを特徴とする請求項7に記載の細胞培養方法。 The cell culture method according to claim 7, characterized in that aligning the cell culture carrier plane on a medium by cutting a bundle of the hollow fibers.
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