JP4186043B2 - Cell differentiation inducing method and cell culture - Google Patents
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【０００１】
【発明の属する技術分野】
本発明は、細胞培養、組織培養等の分野において利用される未分化細胞の分化誘導方法に関する。特に、未分化な細胞を効率良く分化誘導できる未分化細胞の分化誘導方法に関する。
【０００２】
【従来の技術】
近年、ヒトの体内に存在する再生能力を持つ細胞を利用して細胞移植治療を行う再生医療に注目が集まっており、この再生医療により機能不全に陥った生体組織・臓器の機能再生を行なうことが期待されている。
【０００３】
再生医療においては、細胞の分化制御を行なうことが必須であり、細胞の分化機構が破綻すると腫瘍性疾患等の原因となることが知られている。このため、再生医療においては、未分化な細胞群を一定の分化方向に効率良く誘導する分化制御を行う技術が求められている。
【０００４】
従来行われている単層培養（２次元培養）のような細胞培養方法では、種々の細胞を効率良く一定の分化方向に分化誘導することは困難である。例えば軟骨細胞を単層培養する場合、分化誘導がおきる培養条件において培養を実施しても、一部の細胞は軟骨細胞特有の基質を分泌し形態的にも多角形を示すが、その他の多くの細胞は軟骨細胞としての形質を失った繊維芽細胞様の形態を示すことが知られている。
【０００５】
また、容器内のヒト間葉幹細胞に遠心力をかけることによりこの細胞を三次元形態に結合して得られる遠心ペレットに、ある種の軟骨誘導剤又は因子を接触させると、軟骨形成経路に沿って分化することが報告されている（例えば特許文献１参照）。しかし、この方法では、均質な分化機能を有する細胞の集合体を得ることはできない。
【０００６】
【特許文献１】
特表2000-516802号公報（第７頁第12〜14行）
【０００７】
【発明が解決しようとする課題】
本発明は、未分化な細胞を効率良く一定の方向に分化誘導することができる細胞分化誘導方法、均一に分化した細胞培養物又は組織体、及び、この細胞培養物又は組織体を生体に移植する方法を提供することを主目的とする。
【０００８】
【課題を解決するための手段】
前記目的を達成するために本発明者は研究を重ね、未分化細胞に、透過性膜を介して遠心力又は圧力をかけることにより透過性膜に密着した細胞凝集体を形成し、この凝集体を培養することにより、未分化細胞群の全体を、効率良く、一定の方向に均一に分化させることができることを見出した。また、凝集体培養環境に細胞分化を誘導する成分を添加することにより、一層効率よく一定方向への分化を誘導できることを見出した。
【０００９】
ここで、一定の方向に分化することには、以下の▲１▼〜▲３▼の場合が含まれる。
▲１▼ 未分化細胞では同じ方向に分化する場合でもその速度が異なるために結果として異なる分化機能を有する細胞群が形成されるのに対して、細胞群の全体を同じ速度で分化させることができるため結果として均一な分化機能を有する細胞群が形成される場合。
▲２▼ 未分化細胞では異なる方向に分化しているのに対して同じ方向に分化が進行する場合。
▲３▼ ガン化せずに正常な分化方向に向かう場合。
【００１０】
この分化誘導の詳細な機序は完全に明らかではないが、例えば中空糸内に分化ポテンシャルを持つ細胞を高密度に充填し、更に遠心力や圧力を加えることで細胞同士の接触頻度が高まり、その結果として細胞間情報伝達の亢進、細胞位置情報の獲得等の現象が起こり易くなり、細胞が正常に分化する微小環境が整うためであると、本発明者は推察している。
【００１１】
本発明は前記知見に基づき完成されたものであり、以下の各項の細胞分化誘導方法及び細胞培養物又は組織体などを提供する。
【００１２】
項１． 透過性膜からなる中空糸の内腔に１種または複数種の未分化細胞を入れた状態で、この未分化細胞に遠心力又は圧力をかけることにより細胞の凝集体を形成する工程と；この細胞凝集体を培養することにより未分化細胞を分化誘導する工程とを含む細胞分化誘導方法。
【００１３】
項２． 透過性膜からなる中空糸束と中空糸束を覆うシェルとを備える構造物のシェルと中空糸との間隙に１種または複数種の未分化細胞を入れた状態で、この未分化細胞に遠心力又は圧力をかけることにより細胞の凝集体を形成する工程と；この細胞凝集体を培養することにより未分化細胞を分化誘導する工程とを含む細胞分化誘導方法。
【００１４】
項３． 内部に透過性膜を備えた容器の透過性膜上に１種または複数種の未分化細胞を載せた状態で、この未分化細胞に遠心力又は圧力をかけることにより細胞の凝集体を形成する工程と；この細胞凝集体を培養することにより未分化細胞を分化誘導する工程とを含む細胞分化誘導方法。
【００１５】
項４． 凝集体培養工程において、細胞凝集体を細胞分化誘導成分とともに培養する項１、２又は３に記載の細胞分化誘導方法。
【００１６】
項５． 凝集体培養工程において、細胞凝集体を動物生体内に移植した状態で培養する項１、２又は３に記載の細胞分化誘導方法。
【００１７】
項６． 凝集体形成工程において、細胞の種類が同一又は互いに異なる複数の細胞凝集体を形成し、凝集体培養工程において、これら複数の細胞凝集体を同一培養系内で共培養する項１から５のいずれかに記載の細胞分化誘導方法。
【００１８】
項７． 未分化細胞が、胚性幹細胞、外胚葉幹細胞、中胚葉幹細胞、内胚葉幹細胞、間葉系幹細胞、造血幹細胞、神経幹細胞、肝幹細胞、筋幹細胞、膵幹細胞、皮膚幹細胞、網膜幹細胞、毛包幹細胞、骨前駆細胞、脂肪前駆細胞、軟骨細胞、毛母細胞、上皮細胞、血管内皮細胞、平滑筋細胞、ガン細胞、及びこれらの細胞からの分化系譜内の細胞からなる群より選ばれる細胞である項１から６のいずれかに記載の細胞分化誘導方法。
【００１９】
項８． 項１から７のいずれかに記載の方法により得られる細胞培養物又は組織体。
【００２０】
項９． 項８に記載の細胞培養物又は組織体からなる医用生体材料。
【００２１】
項10． 項９に記載の細胞培養物又は組織体を動物生体に移植する細胞培養物又は組織の移植方法。
【００２２】
【発明の実施の形態】
以下、本発明を詳細に説明する。
（１）細胞培養方法
本発明の第１の細胞分化誘導方法は、透過性膜からなる中空糸の内腔に１種または複数種の未分化細胞を入れた状態で、この未分化細胞に遠心力又は圧力をかけることにより細胞の凝集体を形成する工程と；この細胞凝集体を培養することにより未分化細胞を分化誘導する工程とを含む方法である。
【００２３】
本発明の第２の細胞分化誘導方法は、透過性膜からなる中空糸束と中空糸束を覆うシェルとを備える構造物のシェルと中空糸との間隙に１種または複数種の未分化細胞を入れた状態で、この未分化細胞に遠心力又は圧力をかけることにより細胞の凝集体を形成する工程と；この細胞凝集体を培養することにより未分化細胞を分化誘導する工程とを含む方法である。
【００２４】
本発明の第３の細胞分化誘導方法は、内部に透過性膜を備えた容器の透過性膜上に１種または複数種の未分化細胞を載せた状態で、この未分化細胞に遠心力又は圧力をかけることにより細胞の凝集体を形成する工程と；この細胞凝集体を培養することにより未分化細胞を分化誘導する工程とを含む方法である。
【００２５】
未分化細胞
本発明方法において、細胞の分化とは、未分化な細胞から特定機能を有する細胞へと変化していく現象をいう。全く分化していない又は分化レベルが極めて低い未分化細胞（例えば幹細胞）から最終的に機能する細胞が作り出される過程、及び、すでに分化し特定機能を有する細胞が外部からの刺激に応じて新しい機能を獲得する過程のいずれもが分化に含まれる。
【００２６】
本発明方法の対象となる未分化細胞とは、最終分化に至っていない細胞をいう。未分化細胞としては、特に限定はされないが、例えば胚性幹細胞、外胚葉幹細胞、中胚葉幹細胞、内胚葉幹細胞、間葉系幹細胞、造血幹細胞、神経幹細胞、肝幹細胞、筋幹細胞、膵幹細胞、皮膚幹細胞、網膜幹細胞、毛包幹細胞、骨前駆細胞、脂肪前駆細胞、軟骨細胞、毛母細胞、上皮細胞、血管内皮細胞、平滑筋細胞などの細胞及びこれらの細胞の分化系譜内の細胞が挙げられる。
【００２７】
また、分化ポテンシャルを持つ点で、ガン細胞も未分化細胞に含まれる。このようなガン細胞として、例えば、MC3T3-E1細胞(骨芽細胞に分化して骨を作る細胞株)、MC3T3-G2/PA6細胞(脂肪細胞に分化)などが挙げられる。このように、分化途中にあって最終分化に至っていない細胞、すなわち外部からの刺激に応じて新たな機能を獲得し得る細胞も未分化細胞に含まれる。
【００２８】
また細胞の由来は特に限定されないが、動物、特に哺乳動物に由来するものであることが好ましい。哺乳動物の種類は特に限定されず、本発明の未分化細胞としては、ヒト、マウス、ラット等のいずれの動物由来のものも使用できる。特に、ヒト由来の細胞を用いることが好ましい。
【００２９】
未分化細胞は、１種を単独で又は複数種を混合して使用できる。
【００３０】
細胞凝集体
本発明において細胞凝集体とは、分散された細胞による自発的な凝集ではなし得ない程度に、細胞同士が高度に接着した状態又は高頻度に接着した状態の細胞群をいう。
【００３１】
凝集体形成方法
このような細胞凝集体は、具体的には、１種又は複数種の未分化細胞を例えば適当な液体培地又はバッファーなどに懸濁した状態で、細胞の種類に応じて細胞に傷害を与えない範囲の遠心力又は圧力をかけることにより形成することができる。
【００３２】
また、未分化細胞の凝集体を形成する際に、未分化細胞の分化を制御し得る補助的な細胞を混在させた状態で凝集体を形成することができる。このような補助的な細胞は、細胞間の接触によるシグナル伝達や、その細胞が分泌する液性因子等により未分化細胞の分化を制御できると考えられる。
【００３３】
遠心力の大きさは、細胞の種類によっても異なるが、概ね２〜２０００×Ｇ程度、特に４〜５００×Ｇ程度とすることが好ましい。例えばヒト軟骨細胞の場合には、通常１５００×Ｇ以下、特に１０〜４００×Ｇ程度の遠心力をかけることが好ましい。遠心時間は、細胞凝集体を形成できる範囲で適宜設定すればよく特に限定されない。遠心力は、例えば市販の遠心機を使用してかけることができる。
【００３４】
また、圧力の大きさは、細胞の種類によって異なるが、概ね５〜５０ｋｇ重／ｃｍ2程度、特に５〜３５ｋｇ重／ｃｍ2程度、さらに特に１０〜２０ｋｇ重／ｃｍ2程度が好ましい。圧力は、例えば注射器、アスピレーター（水流ポンプ）、電動ポンプなどを用いてかけることができる。圧力は凝集体を形成できる限り陰圧又は陽圧のいずれであってもよい。
【００３５】
本発明の第１の方法により中空糸内に細胞凝集体を形成するにあたっては、例えば注射器などを用いてアダプターを介して複数の中空糸内に一度に細胞懸濁液等を注入することができる。また、例えば特開2001-128660号公報に記載されているような円筒形のシェル内に毛細血管に見立てた多数の中空糸を微小間隔で規則的に配置した細胞培養用モジュールを用い、モジュールの注入口から複数中空糸内に一度に細胞懸濁液等を注入することもできる。
【００３６】
また、本発明の第２の方法により中空糸間に細胞凝集体を形成するにあたっては、例えば特開2001-128660号公報に記載されているような円筒形のシェル内に毛細血管に見立てた多数の中空糸を微小間隔で規則的に配置した細胞培養用モジュールを採用でき、この細胞培養用モジュールの注入口から細胞懸濁液等を注入することができる。
【００３７】
また、本発明の第３の方法により透過性膜上に細胞凝集体を形成するにあたっては、内部に透過性膜を備えた容器のその透過性膜上に、未分化細胞の懸濁液等を載せた状態で細胞に遠心力又は圧力をかけることによりこの透過性膜上に細胞凝集体を形成することができる。このような容器としては、底面の一部又は全部が透過性膜で構成された容器、底面の他に側面の全部又は一部も透過性膜で構成された容器、複数ウェルを有し各ウェルの底面の一部又は全部が透過性膜で構成された容器、複数ウェルを有し各ウェルの底面の他に側面の全部又は一部も透過性膜で構成された容器等を採用できる。具体的には、例えば、複数ウェルを有し各ウェルの底面が透過性膜で構成されたいわゆるカルチャーインサートを採用できる。このようなカルチャーインサートは、例えばメンブレンカルチャーインサート（イワキガラス株式会社製）等の商品名で、６ウェル、12ウェル、24ウェルなどの複数ウェルを有するものが市販されている。
【００３８】
透過性膜
透過性膜は、細胞は通過させないが水、塩類、蛋白質などの培養液成分は通過させる通孔を多数有する膜であり、通常０．１〜５μｍ程度、特に０．２〜３μｍ程度の通孔を有するものであることが好ましい。透過性膜の厚さは、膜の適度の強度及び良好な物質透過性が保たれる範囲であればよく、特に限定されないが、通常１０〜２００μｍ程度、特に２０〜１００μｍ程度のものが好適に採用される。
【００３９】
透過性膜の材料は、細胞毒性を有さず、滅菌、洗浄、培養液との接触などにより変質、分解しない材料であればよく、特に限定されない。例えばセルロース系樹脂、ポリエチレン、ポリプロピレンのようなポリオレフィン、ポリスルホン、ポリエーテルスルホン、フッ素樹脂、ポリカーボネート、アクリル樹脂等からなる透過性膜を利用できる。この他、生分解性樹脂からなる透過性膜を採用することもでき、この場合は、凝集体形成後にその透過性膜ごと生体に移植することができる。
【００４０】
透過性膜からなる中空糸を採用する場合は、中空糸の大きさは、特に限定されないが、内部の細胞に効率よく栄養分を供給するためには、内径が通常２０〜１０００μm程度、特に５０〜５００μm程度、さらに特に１００〜３００μm程度であることが好ましい。前記範囲であれば、中空糸中心部の細胞の物質交換を十分に行えるとともに、実用上十分な数の細胞を入れることができる。中空糸の長さは、特に限定されないが、例えば０．５〜２０ｃｍ程度、特に１〜１０ｃｍ程度とすることができる。
【００４１】
細胞凝集体の培養
＜ in vitro ＞
得られた細胞凝集体を培養するにあたっては、細胞培養用培地中で培養することができる。細胞培養用培地は、特に限定されず、例えばダルベッコの改変イーグル培地、ウィリアムズＥ培地、ハムのＦ−１０培地、Ｆ−１２培地、ＲＰＭＩ−１６４０培地、ＭＣＤＢ１５３培地、１９９培地など従来より細胞培養用基礎培地として知られている培地を使用できる。培地成分を適宜調整することにより、未分化細胞を所望する細胞に分化させることができる。
【００４２】
また、培地には分化誘導成分を添加することができ、これにより一層効率よく、未分化細胞を所望の分化細胞に誘導することができるようになる。分化誘導成分としては、未分化細胞の種類及び目的とする分化細胞の種類に応じて、従来公知の分化誘導成分を加えることができる。
【００４３】
このような成分として、例えば造血幹細胞に対する分化誘導成分であるインターロイキン（IL）、幹細胞増殖因子（SCF）、エリスロポイエチン(EPO)、インターフェロン（IFN）、トロンボポイエチン(TPO)、腫瘍壊死因子（TNF）、コロニー刺激因子(CSF)等が挙げられる。また、間葉系幹細胞から骨への分化誘導因子としては、デキサメタゾン、β-リン酸グリセロール、アスコルビン酸等が挙げられる。間葉系幹細胞から軟骨細胞への分化誘導因子としてはTGF−β（transforming growth factor-β）が好ましい。また、間葉系幹細胞から脂肪細胞への分化誘導成分としてはデキサメタゾン、ホスホジエステラーゼ阻害剤 の１−メチル−３−イソブチルキサンチン（１−methyl−３−isobutylxanthine）、インスリン、インドメタシン等が挙げられる。
【００４４】
培養温度は、未分化細胞及び目的とする分化細胞の種類によって適した温度とすればよい。培養時間は、未分化細胞及び目的とする分化細胞の種類によって異なるが、24時間〜６ヶ月程度とすればよい。
【００４５】
例えば、間葉系幹細胞細胞を軟骨細胞に分化させようとする場合には、培地としてダルベッコの改変イーグル培地を含有する液体培地を用い、分化誘導成分としてTGF-β、インスリン、トランスフェリン及びデキサメタゾン等を適量添加して３７℃程度で２〜４週間程度培養すればよい。
【００４６】
また、間葉系幹細胞を脂肪前駆細胞に分化させようとする場合には、培地としてダルベッコの改変イーグル培地を含有する液体培地を用い、分化誘導成分としてデキサメタゾン、インシュリン及びインドメタシン等を適量添加して培養すればよい。また、間葉系幹細胞を骨格筋細胞に分化させようとする場合には、培地としてダルベッコの改変イーグル培地を含有する液体培地を用い、分化誘導成分として５−アザシチジン等を適量添加して培養すればよい。
【００４７】
また、未分化細胞が分化して細胞Aを経て細胞Bになる場合には、細胞Aにまで分化した後に分化誘導成分を含まない培地に変換することにより、細胞群全体を細胞Aの状態にすることができる。
【００４８】
中空糸内に凝集体を形成した場合は、中空糸ごと液体培地に浸漬して培養すればよい。また、シェル内に中空糸束を備えた構造物の中空糸内に凝集体を形成した場合は、シェル内の中空糸間隙に細胞培養用液体培地を環流することにより培養することもできる。また、このような構造物の中空糸束間に凝集体を形成した場合は、中空糸内を細胞培養用液体培地を環流することにより、培養すればよい。
【００４９】
中空糸を利用する場合は、透過性膜からなる中空糸を介して細胞に効率よく分化に必要な成分、その他の栄養分及び酸素などを供給できるとともに、効率よく老廃物を排出することができる。
【００５０】
また、例えばカルチャーインサートのような透過性膜上に細胞凝集体を形成した場合は、例えばカルチャーインサートをウェルどうしが適合するマルチウェルプレートに装着し、ウェル内に細胞培養用培地を入れた状態で培養することができる。
＜ in situ ・ in vivo ＞
また、細胞凝集体を動物生体内に移植した状態で培養することによっても未分化細胞の凝集体を分化誘導することができる。この場合は、例えば、内部に細胞凝集体が形成された中空糸をマウス、ラットなどの動物の腹腔内や肝臓等の組織の被膜内に移植し、２４時間程度以上この動物を飼育すればよい。これにより、未分化細胞凝集体を分化細胞に誘導できる。
【００５１】
細胞凝集体を特定組織の近辺に移植することにより、その近辺にある(組織)細胞が放出する分化因子や、凝集体が接する細胞から受け取るシグナルにより、その特定組織の細胞へと分化誘導できる。例えば、未分化な神経細胞の凝集体を脊髄内に移植する場合には神経繊維が形成される。
【００５２】
この他、細胞凝集体を生体内(in vivo)に移植することによりin vitroでは再現できないような分化環境が得られる。この場合は必ずしも、分化により得ようとする組織と同じ組織の近辺に凝集体を移植しなくてもよい。たとえば、軟骨に分化するポテンシャルをもつ未分化細胞を筋肉組織の付近に移植することにより軟骨に分化する。
＜共培養＞
いずれの培養方法を採用する場合も、同一培養系内で細胞凝集体を別の細胞とともに培養することができる。例えば細胞Aを分化させようとする場合に、細胞Aの凝集体とその分化を誘導する因子を放出する細胞Bとを同じ培養容器内で培養することにより、細胞Ａの分化が促進され得る。この場合、細胞Bは、例えば中空糸内に充填されていてもよく、又は、透過性膜を介さずに容器内で培養してもよい。細胞Bを透過性膜を介さずに培養する場合は、例えば容器底面に単層培養すればよい。細胞Bは細胞凝集体を形成していてもよい。
（２）細胞培養物又は組織体
前述した本発明の細胞培養方法により、一定方向に均一に分化した細胞群である細胞培養物又は組織体が得られる。
【００５３】
この細胞培養物又は組織体は、医用生体材料として利用できる。特に、失われた組織、臓器などを代替する材料として好適に利用できる。具体的には、例えば人工肝臓、人工膵臓、人工腎臓、人工心臓のような人工臓器；人工消化管；人工血管；人工皮膚、人工神経；人工骨；人工軟骨；人工内耳；人工水晶体；人工角膜、人工脂肪などとして利用できる。
【００５４】
例えば内部に多数の中空糸束を備えた細胞培養用モジュールの中空糸内で肝幹細胞の凝集体を培養することより肝細胞群を得た場合は、肝細胞群が充填されたモジュールをそのまま人工肝臓として用いることができる。このモジュールは、例えば血液の体外循環系に組み入れることによって体外人工肝臓として利用できる。
（３）移植方法
本発明の移植方法は、上記説明した本発明の細胞培養物又は組織体を動物生体に移植する方法である。例えば透過性膜からなる中空糸内に未分化細胞を遠心力を利用して充填して凝集体とし、この細胞凝集体が充填された中空糸を動物生体内に移植すればよい。例えば中空糸内に形成した脂肪前駆細胞の凝集体を、中空糸ごと腹腔内に移植することにより効率良く脂肪細胞に分化させることができる。あるいは、生分解性をもつ透過性膜からなる中空糸内に充填して凝集体とした神経幹細胞を、中空糸ごと脊髄内に移植することにより神経繊維の形成を期待することができる。この方法は脊髄損傷の有功な治療法となると考えられる。
【００５５】
【実施例】
以下、実施例及び試験例を示して本発明をより詳細に説明するが、本発明はこれらの実施例に限定されるものではない。
【００５６】
実施例１
フラスコにて培養した初代ヒト軟骨細胞をトリプシン消化し、７．５×１０6個／ｍｌの細胞分散液を調製した。これをセルローストリアセテート製の血漿分離用中空糸（東洋紡績社製ＡＰ２５０Ｎ１５タイプ；内径２８５μｍ、外径３８７μｍ）内腔に注入した後、６０×Ｇの遠心力を９０秒間加えることにより、中空糸内腔に細胞凝集体を形成させた。
【００５７】
軟骨細胞の凝集体が封入された状態の長さ３cmの中空糸６本を作成し、直径３５ｍｍの培養ディッシュ（ファルコン製）に入れ、Dulbecco's modified eagle medium（ＤＭＥＭ；ギブコ製）に、ウシ胎児血清５％、インシュリン（シグマ製）、ＴＧＦ−β、トランスフェリン、ペニシリンＧカリウム（ナカライテスク製）、ストレプトマイシン硫酸塩（ナカライテスク製）、炭酸水素ナトリウム（ナカライテスク製）を添加した分化誘導培地にて、５％炭酸ガス、９５％大気の雰囲気下、振盪器上で３７℃で１ヶ月間の振盪培養を行い分化誘導を行なった。
【００５８】
中空糸内の細胞を経日的にホルマリン固定およびパラフィン包埋し、薄切片を作製しトルイジンブルー染色を実施することにより細胞の分化の様子を調べた。
【００５９】
比較例１
実施例１において調製したヒト軟骨細胞の分散液を分化誘導培地にて０．２×１０6個／ｍｌになるよう希釈し、培養面積が２５ｃｍ2のフラスコ（ファルコン社製）に播種し、実施例１で用いたのと同様の分化誘導培地５ｍｌを入れ実施例１と同じ環境下にて単層培養を行った。経日的に細胞をメタノール固定し、トルイジンブルー染色を行ない顕微鏡で観察した。
【００６０】
実施例１及び比較例１により観察された細胞のトルイジンブルー染色像をそれぞれ図１及び図２に示す。図１及び図２において、（Ａ）は培養開始１週間目の様子を示し、（Ｂ）は培養開始２週間目の様子を示し、培養開始４週間目の様子を示す。
【００６１】
図１から明らかなように、実施例１により得られた中空糸内の軟骨細胞培養物は分化誘導培養開始後１週間目（Ａ）には軟骨基質形成を示すトルイジンブルーによる異染性が認められないが、培養開始後２週間目（Ｂ）より細胞に異染性が認められるようになり、培養開始後４週間目（Ｃ）には細胞組織体全体にわたり異染性が顕著に認められた。培養開始４週間目には、組織体は均質な分化機能をもった細胞群から形成されていると考えられる。
【００６２】
実施例１では、最終分化していない即ち分化ポテンシャルを有する軟骨細胞が、本発明の分化誘導法により最終分化形態である軟骨組織形成へと近づいた。
【００６３】
これに対し比較例１の単層培養により得られた軟骨細胞培養物は、実施例１と同様に培養開始後１週間目（Ａ）にはトルイジンブルーによる異染性を示していないが、培養開始後２週間目（Ｂ）より細胞に異染性が認められるようになり、培養開始後４週間目（Ｃ）には異染性を示す細胞が認められた。しかし、培養開始後４週間目においても、異染性を示す細胞は細胞密度の高い部分に限定されており、培養された細胞全体が分化誘導されることはなかった。
実施例２
フラスコにて培養した初代ヒト前駆脂肪細胞をトリプシン消化し、７．５×１０6個／ｍｌの細胞分散液を調製した。これをセルローストリアセテート製の血漿分離用中空糸（東洋紡績社製ＡＰ２５０Ｎ１５タイプ；内径２８５μｍ、外径３８７μｍ）内腔に注入した後、６０×Ｇの遠心力を９０秒間加えることにより、中空糸内腔に細胞凝集体を形成させた。
【００６４】
ヒト前駆脂肪細胞の凝集体が封入された状態の長さ３cmの中空糸６本を作成し、直径３５ｍｍの培養ディッシュ（ファルコン製）に入れ、Dulbecco's modified eagle medium（ＤＭＥＭ）に、ウシ胎児血清２％、インシュリン、トランスフェリン、トリヨードサイロニン、上皮成長因子、デキサメタゾン、インドメタシン、ペニシリンＧカリウム、ストレプトマイシン硫酸塩、炭酸水素ナトリウムを添加した分化誘導培地にて、５％炭酸ガス、９５％大気の雰囲気下、振盪器上で３７℃で振盪培養を行い分化誘導を行なった。２週間培養後、中空糸をＰＢＳ（phosphate buffer-saline）で洗浄し、中性脂肪測定用診断薬にて細胞内に蓄積した中性脂肪量を測定した。
【００６５】
比較例２
実施例２において調製したヒト前駆脂肪細胞の分散液を分化誘導培地にて０．２×１０6個／ｍｌになるよう希釈し、直径３５ｍｍの培養ディッシュに播種し、実施例２で用いたのと同様の分化誘導培地５ｍｌを入れ実施例１と同じ環境下にて単層培養を行った。２週間培養後、細胞をＰＢＳで洗浄し、中性脂肪測定用診断薬にて細胞内に蓄積した中性脂肪量を測定した。
【００６６】
実施例２及び比較例２における細胞内の中性脂肪蓄積量を図３に示す。図３から明らかなように、実施例２においては、細胞内に蓄積された中性脂肪量は比較例２のそれに比べ、著しく多いものであった。このことから、本発明の分化誘導法により前駆脂肪細胞から脂肪細胞への分化が効率よく行われたことが示唆された。
【００６７】
【発明の効果】
本発明によれば、未分化な細胞を効率良く一定の方向に分化誘導することができる細胞分化誘導方法、均一に分化した細胞培養物又は組織体、及び、この細胞培養物又は組織体を生体に移植する方法を提供することができる。
【００６８】
従来の単層培養法により未分化細胞を培養する場合は、細胞群の全体を一定の方向に分化誘導することが困難で、すなわち一部の細胞が分化して一部の細胞が未分化のまま残ったり、互いに異なる機能を備える複数種の細胞の混合物となったり、細胞が正常に分化しなかったりする。この点、本発明方法では、未分化細胞に遠心力又は圧力をかけることにより透過性膜上に細胞の凝集体を形成し、この凝集体を培養することにより、未分化細胞群の全体を、効率良く、一定の方向に均一に分化させることができる。
【００６９】
さらに、凝集体培養環境に細胞分化を誘導する成分を添加することにより、一層効率よく一定方向への分化を誘導できる。
【００７０】
また、本発明方法により、均質な分化機能を持った３次元構造の細胞組織体が簡便に効率良く得られる。
【図面の簡単な説明】
【図１】本発明の分化誘導方法の１実施例において分化誘導することにより得られた軟骨細胞培養物のトルイジンブルー染色像である。
【図２】従来の単層培養法で分化誘導することにより得られた軟骨細胞培養物のトルイジンブルー染色像である。
【図３】実施例２及び比較例２において得られた細胞内蓄積中性脂肪量を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for inducing differentiation of undifferentiated cells used in fields such as cell culture and tissue culture. In particular, the present invention relates to a method for inducing differentiation of undifferentiated cells that can efficiently induce differentiation of undifferentiated cells.
[0002]
[Prior art]
In recent years, regenerative medicine that performs cell transplantation treatment using cells with regenerative capacity existing in the human body has attracted attention, and functional regeneration of living tissues and organs that have malfunctioned due to this regenerative medicine Is expected.
[0003]
In regenerative medicine, it is essential to control the differentiation of cells, and it is known that if the cell differentiation mechanism breaks down, it may cause a neoplastic disease or the like. Therefore, in regenerative medicine, a technique for performing differentiation control that efficiently induces undifferentiated cell groups in a certain differentiation direction is required.
[0004]
In conventional cell culture methods such as monolayer culture (two-dimensional culture), it is difficult to efficiently induce differentiation of various cells in a certain differentiation direction. For example, when chondrocytes are cultured in a monolayer, some cells secrete a chondrocyte-specific substrate and show a polygonal shape even if they are cultured under culture conditions that induce differentiation. These cells are known to show a fibroblast-like morphology that has lost its trait as a chondrocyte.
[0005]
In addition, when a certain cartilage inducer or factor is brought into contact with a centrifugal pellet obtained by binding a human mesenchymal stem cell in a container to a three-dimensional form by applying a centrifugal force, it follows the chondrogenic pathway. Has been reported to differentiate (see, for example, Patent Document 1). However, this method cannot provide an aggregate of cells having a homogeneous differentiation function.
[0006]
[Patent Document 1]
Special Table 2000-516802 Publication (page 7, lines 12-14)
[0007]
[Problems to be solved by the invention]
The present invention relates to a cell differentiation induction method capable of efficiently inducing differentiation of undifferentiated cells in a specific direction, a uniformly differentiated cell culture or tissue body, and transplanting the cell culture or tissue body into a living body. The main purpose is to provide a way to do this.
[0008]
[Means for Solving the Problems]
In order to achieve the above-mentioned object, the present inventor has repeatedly researched to form a cell aggregate closely adhered to the permeable membrane by applying centrifugal force or pressure to the undifferentiated cell through the permeable membrane. It has been found that the whole undifferentiated cell group can be efficiently and uniformly differentiated in a certain direction by culturing. It was also found that differentiation in a certain direction can be induced more efficiently by adding a component that induces cell differentiation to the aggregate culture environment.
[0009]
Here, differentiating in a certain direction includes the following cases (1) to (3).
(1) In the case of undifferentiated cells, even when they differentiate in the same direction, their speeds are different. As a result, cell groups having different differentiation functions are formed. On the other hand, the whole cell group can be differentiated at the same speed. As a result, a cell group having a uniform differentiation function is formed.
(2) When undifferentiated cells differentiate in different directions, whereas differentiation progresses in the same direction.
(3) When heading in the normal differentiation direction without becoming cancerous.
[0010]
Although the detailed mechanism of this differentiation induction is not completely clear, for example, cells with differentiation potential are packed in a hollow fiber at a high density, and further, the frequency of contact between cells increases by applying centrifugal force or pressure, As a result, the present inventor speculates that phenomena such as enhancement of cell-to-cell information transmission and acquisition of cell position information are likely to occur, and a microenvironment in which cells normally differentiate is prepared.
[0011]
The present invention has been completed based on the above findings, and provides the following methods for inducing cell differentiation and cell cultures or tissues.
[0012]
Item 1. Forming a cell aggregate by applying centrifugal force or pressure to the undifferentiated cells in a state where one or a plurality of undifferentiated cells are put in the lumen of a hollow fiber made of a permeable membrane; And a method for inducing differentiation of undifferentiated cells by culturing cell aggregates.
[0013]
Item 2. Centrifugation is performed on the undifferentiated cells in a state where one or more types of undifferentiated cells are put in the gap between the shell and the hollow fiber of the structure including a hollow fiber bundle made of a permeable membrane and a shell covering the hollow fiber bundle. A method for inducing cell differentiation comprising a step of forming an aggregate of cells by applying force or pressure; and a step of inducing differentiation of undifferentiated cells by culturing the cell aggregate.
[0014]
Item 3. In a state where one or a plurality of types of undifferentiated cells are placed on a permeable membrane of a container having a permeable membrane therein, a cell aggregate is formed by applying centrifugal force or pressure to the undifferentiated cells. A method for inducing cell differentiation comprising: a step; and a step of inducing differentiation of an undifferentiated cell by culturing the cell aggregate.
[0015]
Item 4. Item 4. The method for inducing cell differentiation according to Item 1, 2, or 3, wherein the cell aggregate is cultured together with a cell differentiation-inducing component in the aggregate culture step.
[0016]
Item 5. Item 4. The method for inducing cell differentiation according to Item 1, 2 or 3, wherein in the aggregate culturing step, the cell aggregate is cultured in a state of being transplanted into an animal body.
[0017]
Item 6. Any one of Items 1 to 5, wherein a plurality of cell aggregates having the same or different cell types are formed in the aggregate formation step, and the plurality of cell aggregates are co-cultured in the same culture system in the aggregate culture step. The method for inducing cell differentiation according to claim 1.
[0018]
Item 7. Undifferentiated cells are embryonic stem cells, ectoderm stem cells, mesoderm stem cells, endoderm stem cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells, muscle stem cells, pancreatic stem cells, skin stem cells, retinal stem cells, hair follicle stem cells A cell selected from the group consisting of bone precursor cells, adipose precursor cells, chondrocytes, hair matrix cells, epithelial cells, vascular endothelial cells, smooth muscle cells, cancer cells, and cells within the differentiation lineage from these cells Item 7. The method for inducing cell differentiation according to any one of Items 1 to 6.
[0019]
Item 8. Item 8. A cell culture or tissue obtained by the method according to any one of Items 1 to 7.
[0020]
Item 9. Item 9. A medical biomaterial comprising the cell culture or tissue according to Item 8.
[0021]
Item 10. Item 10. A cell culture or tissue transplantation method of transplanting the cell culture or tissue body according to Item 9 into an animal body.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
(1) Cell culture method
In the first method for inducing cell differentiation of the present invention, centrifugal force or pressure is applied to an undifferentiated cell in a state where one or more kinds of undifferentiated cells are put in the lumen of a hollow fiber made of a permeable membrane. And a step of inducing differentiation of an undifferentiated cell by culturing the cell aggregate.
[0023]
In the second cell differentiation inducing method of the present invention, one or a plurality of types of undifferentiated cells are provided in a gap between a shell of a structure including a hollow fiber bundle made of a permeable membrane and a shell covering the hollow fiber bundle and the hollow fiber. A step of forming an aggregate of cells by applying centrifugal force or pressure to the undifferentiated cells in a state of putting a cell; and a step of inducing differentiation of the undifferentiated cells by culturing the cell aggregate. It is.
[0024]
In the third method for inducing cell differentiation of the present invention, in the state where one or a plurality of types of undifferentiated cells are placed on a permeable membrane of a container having a permeable membrane therein, centrifugal force or The method includes a step of forming an aggregate of cells by applying pressure; and a step of inducing differentiation of an undifferentiated cell by culturing the cell aggregate.
[0025]
Undifferentiated cells
In the method of the present invention, cell differentiation refers to a phenomenon that changes from an undifferentiated cell to a cell having a specific function. The process by which cells that ultimately function from undifferentiated cells (for example, stem cells) that are not differentiated at all or have a very low differentiation level are created, and cells that have already differentiated and have a specific function have new functions in response to external stimuli Any of the processes of acquiring is included in differentiation.
[0026]
The undifferentiated cell to be subjected to the method of the present invention refers to a cell that has not reached final differentiation. Examples of undifferentiated cells include, but are not limited to, embryonic stem cells, ectoderm stem cells, mesoderm stem cells, endoderm stem cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells, muscle stem cells, pancreatic stem cells, skin Examples include stem cells, retinal stem cells, hair follicle stem cells, osteoprogenitor cells, adipose precursor cells, chondrocytes, hair matrix cells, epithelial cells, vascular endothelial cells, smooth muscle cells, and cells in the differentiation lineage of these cells. .
[0027]
Cancer cells are also included in undifferentiated cells in that they have a differentiation potential. Examples of such cancer cells include MC3T3-E1 cells (cell lines that differentiate into osteoblasts to form bone), MC3T3-G2 / PA6 cells (differentiate into adipocytes), and the like. Thus, cells that are in the middle of differentiation and have not yet reached final differentiation, that is, cells that can acquire a new function in response to an external stimulus are also included in the undifferentiated cells.
[0028]
The origin of the cell is not particularly limited, but is preferably derived from an animal, particularly a mammal. The type of mammal is not particularly limited, and as the undifferentiated cell of the present invention, those derived from any animal such as human, mouse, rat and the like can be used. In particular, it is preferable to use human-derived cells.
[0029]
An undifferentiated cell can be used individually by 1 type or in mixture of multiple types.
[0030]
Cell aggregate
In the present invention, the cell aggregate refers to a cell group in a state where the cells are highly adhered or frequently adhered to such an extent that spontaneous aggregation by dispersed cells cannot be achieved.
[0031]
Aggregate formation method
Specifically, such cell aggregates do not damage cells depending on the type of cells in a state where one or more types of undifferentiated cells are suspended in, for example, an appropriate liquid medium or buffer. It can be formed by applying a range of centrifugal force or pressure.
[0032]
Moreover, when forming an aggregate of undifferentiated cells, the aggregate can be formed in a state where auxiliary cells capable of controlling the differentiation of undifferentiated cells are mixed. Such auxiliary cells are thought to be able to control the differentiation of undifferentiated cells by signal transduction by contact between cells, humoral factors secreted by the cells, and the like.
[0033]
The magnitude of the centrifugal force varies depending on the type of cell, but is preferably about 2 to 2000 × G, particularly about 4 to 500 × G. For example, in the case of human chondrocytes, it is preferable to apply a centrifugal force of usually 1500 × G or less, particularly about 10 to 400 × G. The centrifugation time is not particularly limited as long as it is set as appropriate as long as cell aggregates can be formed. The centrifugal force can be applied using, for example, a commercially available centrifuge.
[0034]
Moreover, although the magnitude | size of a pressure changes with kinds of cell, it is about 5-50 kg weight / cm in general.2Degree, especially 5-35kg weight / cm2Degree, more particularly 10-20 kg weight / cm2The degree is preferred. The pressure can be applied using, for example, a syringe, an aspirator (water flow pump), an electric pump, or the like. The pressure may be either negative or positive as long as an aggregate can be formed.
[0035]
In forming a cell aggregate in a hollow fiber by the first method of the present invention, a cell suspension or the like can be injected into a plurality of hollow fibers at once via an adapter using, for example, a syringe. . Further, for example, a module for cell culture in which a large number of hollow fibers arranged like capillary blood vessels are regularly arranged at minute intervals in a cylindrical shell as described in JP-A-2001-128660 is used. It is also possible to inject a cell suspension or the like into a plurality of hollow fibers from the injection port at a time.
[0036]
In forming cell aggregates between hollow fibers by the second method of the present invention, a large number of capillaries are considered in a cylindrical shell as described in, for example, JP-A-2001-128660. It is possible to employ a cell culture module in which the hollow fibers are regularly arranged at minute intervals, and a cell suspension or the like can be injected from the inlet of the cell culture module.
[0037]
In addition, when the cell aggregate is formed on the permeable membrane by the third method of the present invention, an undifferentiated cell suspension or the like is placed on the permeable membrane of the container having the permeable membrane inside. Cell aggregates can be formed on the permeable membrane by applying centrifugal force or pressure to the cells in the loaded state. Examples of such a container include a container in which a part or all of the bottom surface is made of a permeable membrane, a container in which all or part of the side surface is also made of a permeable membrane in addition to the bottom surface, and each well having a plurality of wells. A container in which a part or all of the bottom surface is made of a permeable membrane, a container having a plurality of wells, and in addition to the bottom surface of each well, all or a part of the side surface is also made of a permeable membrane can be adopted. Specifically, for example, a so-called culture insert in which a plurality of wells are provided and the bottom surface of each well is formed of a permeable membrane can be employed. Such a culture insert has a commercial name such as a membrane culture insert (manufactured by Iwaki Glass Co., Ltd.) and has a plurality of wells such as 6 wells, 12 wells, and 24 wells.
[0038]
Permeable membrane
The permeable membrane is a membrane having a large number of through-holes through which cells do not pass but through which components of culture solution such as water, salts and proteins pass, and is usually about 0.1 to 5 μm, particularly about 0.2 to 3 μm. It is preferable that it has. The thickness of the permeable membrane is not particularly limited as long as the moderate strength and good substance permeability of the membrane can be maintained, but a thickness of about 10 to 200 μm, particularly about 20 to 100 μm is preferable. Adopted.
[0039]
The material of the permeable membrane is not particularly limited as long as it does not have cytotoxicity and does not change or decompose by sterilization, washing, contact with a culture solution, or the like. For example, a permeable membrane made of cellulose resin, polyolefin such as polyethylene or polypropylene, polysulfone, polyethersulfone, fluororesin, polycarbonate, acrylic resin or the like can be used. In addition, a permeable membrane made of a biodegradable resin can also be employed. In this case, the permeable membrane can be transplanted into the living body after the aggregate is formed.
[0040]
When employing a hollow fiber made of a permeable membrane, the size of the hollow fiber is not particularly limited, but in order to efficiently supply nutrients to the cells inside, the inner diameter is usually about 20 to 1000 μm, particularly 50 to 50 μm. It is preferably about 500 μm, more preferably about 100 to 300 μm. If it is the said range, while the substance exchange of the cell of a hollow fiber center part can fully be performed, a practically sufficient number of cells can be put. The length of the hollow fiber is not particularly limited, but can be, for example, about 0.5 to 20 cm, particularly about 1 to 10 cm.
[0041]
Cell aggregate culture
< in vitro >
In culturing the obtained cell aggregate, it can be cultured in a cell culture medium. The medium for cell culture is not particularly limited. For example, Dulbecco's modified Eagle medium, Williams E medium, Ham's F-10 medium, F-12 medium, RPMI-1640 medium, MCDB153 medium, 199 medium, etc. Media known as basal media can be used. By appropriately adjusting the medium components, undifferentiated cells can be differentiated into desired cells.
[0042]
In addition, a differentiation-inducing component can be added to the medium, which makes it possible to induce undifferentiated cells into desired differentiated cells more efficiently. As a differentiation-inducing component, a conventionally known differentiation-inducing component can be added depending on the type of undifferentiated cells and the type of targeted differentiated cells.
[0043]
Examples of such components include interleukin (IL), stem cell growth factor (SCF), erythropoietin (EPO), interferon (IFN), thrombopoietin (TPO), and tumor necrosis factor, which are differentiation-inducing components for hematopoietic stem cells. (TNF), colony stimulating factor (CSF) and the like. Examples of the differentiation inducing factor from mesenchymal stem cells to bone include dexamethasone, β-glycerol phosphate, ascorbic acid and the like. As a differentiation inducing factor from mesenchymal stem cells to chondrocytes, TGF-β (transforming growth factor-β) is preferable. Examples of components that induce differentiation from mesenchymal stem cells to adipocytes include dexamethasone, phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine, insulin, indomethacin, and the like.
[0044]
The culture temperature may be a temperature suitable for the type of undifferentiated cells and the desired differentiated cells. The culture time varies depending on the types of undifferentiated cells and target differentiated cells, but may be about 24 hours to 6 months.
[0045]
For example, when a mesenchymal stem cell is to be differentiated into a chondrocyte, a liquid medium containing Dulbecco's modified Eagle medium is used as a medium, and TGF-β, insulin, transferrin, dexamethasone, etc. are used as differentiation-inducing components. Appropriate amount may be added and cultured at about 37 ° C. for about 2 to 4 weeks.
[0046]
When attempting to differentiate mesenchymal stem cells into preadipocytes, use a liquid medium containing Dulbecco's modified Eagle medium as the medium, and add appropriate amounts of dexamethasone, insulin, indomethacin, etc. as differentiation-inducing components. What is necessary is just to culture. When the mesenchymal stem cells are to be differentiated into skeletal muscle cells, use a liquid medium containing Dulbecco's modified Eagle medium as the medium and add an appropriate amount of 5-azacytidine as a differentiation-inducing component. That's fine.
[0047]
In addition, when an undifferentiated cell differentiates and becomes cell B via cell A, it is differentiated to cell A and then converted to a medium containing no differentiation-inducing component, so that the entire cell group is brought into the state of cell A. can do.
[0048]
When aggregates are formed in the hollow fibers, the hollow fibers may be immersed in a liquid medium and cultured. In addition, when an aggregate is formed in the hollow fiber of a structure having a hollow fiber bundle in the shell, it can be cultured by circulating a cell culture liquid medium through the hollow fiber gap in the shell. In addition, when an aggregate is formed between the hollow fiber bundles of such a structure, the culture may be performed by circulating a cell culture liquid medium through the hollow fiber.
[0049]
When the hollow fiber is used, components necessary for differentiation, other nutrients, oxygen, and the like can be efficiently supplied to the cells through the hollow fiber made of a permeable membrane, and waste products can be efficiently discharged.
[0050]
For example, when cell aggregates are formed on a permeable membrane such as a culture insert, for example, the culture insert is mounted on a multi-well plate that fits wells, and a cell culture medium is placed in the well. It can be cultured.
< in situ ・ in vivo >
Further, the differentiation of undifferentiated cell aggregates can also be induced by culturing the cell aggregates in a state of being transplanted into an animal body. In this case, for example, hollow fibers in which cell aggregates are formed can be transplanted into the abdominal cavity of an animal such as a mouse or rat, or a tissue coating such as a liver, and the animal can be raised for about 24 hours or longer. . Thereby, an undifferentiated cell aggregate can be induced | guided | derived to a differentiated cell.
[0051]
By transplanting a cell aggregate in the vicinity of a specific tissue, differentiation can be induced into a cell of the specific tissue by a differentiation factor released by a (tissue) cell in the vicinity or a signal received from a cell in contact with the aggregate. For example, nerve fibers are formed when an aggregate of undifferentiated nerve cells is transplanted into the spinal cord.
[0052]
In addition, a differentiation environment that cannot be reproduced in vitro can be obtained by transplanting cell aggregates in vivo. In this case, the aggregate does not necessarily have to be transplanted in the vicinity of the same tissue as the tissue to be obtained by differentiation. For example, it differentiates into cartilage by transplanting undifferentiated cells having the potential to differentiate into cartilage in the vicinity of muscle tissue.
<Co-culture>
Whichever culture method is employed, the cell aggregate can be cultured with other cells in the same culture system. For example, when the cell A is to be differentiated, the differentiation of the cell A can be promoted by culturing the aggregate of the cell A and the cell B releasing the factor inducing the differentiation in the same culture vessel. In this case, the cell B may be filled in, for example, a hollow fiber, or may be cultured in a container without using a permeable membrane. When culturing cells B without a permeable membrane, for example, monolayer culture may be performed on the bottom surface of the container. Cell B may form a cell aggregate.
(2) Cell culture or tissue
By the above-described cell culture method of the present invention, a cell culture or tissue body which is a group of cells uniformly differentiated in a certain direction can be obtained.
[0053]
This cell culture or tissue can be used as a biomedical material. In particular, it can be suitably used as a material for replacing lost tissues, organs, and the like. Specifically, artificial organs such as artificial liver, artificial pancreas, artificial kidney, and artificial heart; artificial digestive tract; artificial blood vessel; artificial skin, artificial nerve; artificial bone; artificial cartilage; artificial inner ear; artificial crystalline lens; It can be used as artificial fat.
[0054]
For example, when a hepatocyte group is obtained by culturing an aggregate of hepatic stem cells in the hollow fiber of a cell culture module having a large number of hollow fiber bundles inside, the module filled with the hepatocyte group is artificially used as it is. Can be used as liver. This module can be used as an extracorporeal artificial liver, for example, by incorporating it into the extracorporeal circulation system of blood.
(3) Transplantation method
The transplantation method of the present invention is a method of transplanting the above-described cell culture or tissue of the present invention into an animal body. For example, undifferentiated cells may be filled into a hollow fiber made of a permeable membrane using centrifugal force to form an aggregate, and the hollow fiber filled with the cell aggregate may be transplanted into an animal body. For example, agglomerates of preadipocytes formed in the hollow fiber can be efficiently differentiated into adipocytes by transplanting the whole hollow fiber into the abdominal cavity. Alternatively, the formation of nerve fibers can be expected by transplanting the nerve stem cells, which are filled into hollow fibers made of a biodegradable permeable membrane into aggregates, into the spinal cord together with the hollow fibers. This method is considered to be an effective treatment for spinal cord injury.
[0055]
【Example】
EXAMPLES Hereinafter, although an Example and a test example are shown and this invention is demonstrated in detail, this invention is not limited to these Examples.
[0056]
Example 1
The primary human chondrocytes cultured in the flask were digested with trypsin to obtain 7.5 × 106Cells / ml of cell dispersion was prepared. This was injected into a hollow fiber for plasma separation made of cellulose triacetate (AP250N15 type manufactured by Toyobo Co., Ltd .; inner diameter 285 μm, outer diameter 387 μm), and then a centrifugal force of 60 × G was applied for 90 seconds to obtain a hollow fiber lumen. Cell aggregates were formed.
[0057]
Six hollow fibers with a length of 3 cm in which chondrocyte aggregates are encapsulated are prepared, placed in a culture dish (made by Falcon) having a diameter of 35 mm, and fetal bovine serum in Dulbecco's modified eagle medium (DMEM; made by Gibco). 5%, differentiation induction medium supplemented with insulin (manufactured by Sigma), TGF-β, transferrin, penicillin G potassium (manufactured by Nacalai Tesque), streptomycin sulfate (manufactured by Nacalai Tesque), sodium hydrogen carbonate (manufactured by Nacalai Tesque) Differentiation induction was performed by shaking culture at 37 ° C. for 1 month on a shaker in an atmosphere of 5% carbon dioxide gas and 95% air.
[0058]
Cells in the hollow fiber were fixed in formalin and paraffin embedded daily, thin sections were prepared, and toluidine blue staining was performed to examine the state of cell differentiation.
[0059]
Comparative Example 1
The human chondrocyte dispersion prepared in Example 1 was 0.2 × 10 6 in differentiation induction medium.6The culture area is 25 cm.2And 5 ml of the differentiation induction medium similar to that used in Example 1 was added, and monolayer culture was performed in the same environment as in Example 1. The cells were fixed with methanol over time, stained with toluidine blue, and observed with a microscope.
[0060]
The toluidine blue stained images of the cells observed in Example 1 and Comparative Example 1 are shown in FIGS. 1 and 2, respectively. 1 and 2, (A) shows the state of the first week of culture, (B) shows the state of the second week of culture, and shows the state of the fourth week of culture.
[0061]
As is clear from FIG. 1, the chondrocyte culture in the hollow fiber obtained in Example 1 was found to be metachromatic with toluidine blue indicating cartilage matrix formation in the first week (A) after the start of differentiation induction culture. However, from the 2nd week (B) after the start of the culture, the cells were found to be metachromatic, and at the 4th week (C) after the start of the culture, the metachromatics were remarkably observed throughout the cell tissue. It was. At 4 weeks from the start of the culture, the tissue is considered to be formed from a group of cells having a homogeneous differentiation function.
[0062]
In Example 1, chondrocytes that were not terminally differentiated, that is, had differentiation potential, approached the formation of cartilage tissue, which is the final differentiated form, by the differentiation induction method of the present invention.
[0063]
On the other hand, the chondrocyte culture obtained by the monolayer culture of Comparative Example 1 does not show metachromaticity with toluidine blue in the first week (A) after the start of culture, as in Example 1. From the 2nd week (B) after the start, the cells were found to be metachromatic, and at 4 weeks (C) after the start of the culture, cells showing the metachromatic properties were observed. However, even in the fourth week after the start of the culture, the cells showing metachromaticity were limited to a portion having a high cell density, and the whole cultured cells were not induced to differentiate.
Example 2
The primary human preadipocytes cultured in flasks were digested with trypsin to obtain 7.5 × 10 56Cells / ml of cell dispersion was prepared. This was injected into a hollow fiber for plasma separation made of cellulose triacetate (AP250N15 type manufactured by Toyobo Co., Ltd .; inner diameter 285 μm, outer diameter 387 μm), and then a centrifugal force of 60 × G was applied for 90 seconds to obtain a hollow fiber lumen. Cell aggregates were formed.
[0064]
Six hollow fibers with a length of 3 cm, in which human preadipocytes aggregates are encapsulated, are prepared, placed in a 35 mm diameter culture dish (Falcon), and placed in Dulbecco's modified eagle medium (DMEM) with fetal bovine serum 2 %, Insulin, transferrin, triiodothyronine, epidermal growth factor, dexamethasone, indomethacin, penicillin G potassium, streptomycin sulfate, sodium bicarbonate in a differentiation-inducing medium, 5% carbon dioxide, 95% atmosphere Then, differentiation was induced by shaking culture at 37 ° C. on a shaker. After culturing for 2 weeks, the hollow fiber was washed with PBS (phosphate buffer-saline), and the amount of neutral fat accumulated in the cells was measured with a diagnostic agent for measuring neutral fat.
[0065]
Comparative Example 2
The human preadipocyte dispersion prepared in Example 2 was 0.2 × 10 6 in differentiation induction medium.6The solution was diluted so that the number of cells / ml was inoculated, seeded in a culture dish having a diameter of 35 mm, and 5 ml of the differentiation induction medium similar to that used in Example 2 was added, and monolayer culture was performed in the same environment as in Example 1. After culturing for 2 weeks, the cells were washed with PBS, and the amount of neutral fat accumulated in the cells was measured with a diagnostic agent for measuring neutral fat.
[0066]
The intracellular triglyceride accumulation in Example 2 and Comparative Example 2 is shown in FIG. As apparent from FIG. 3, in Example 2, the amount of neutral fat accumulated in the cells was significantly larger than that in Comparative Example 2. This suggested that differentiation from preadipocytes to adipocytes was efficiently performed by the differentiation induction method of the present invention.
[0067]
【The invention's effect】
According to the present invention, a cell differentiation induction method capable of efficiently inducing differentiation of undifferentiated cells in a certain direction, a uniformly differentiated cell culture or tissue body, and the cell culture or tissue body in a living body. A method of transplanting can be provided.
[0068]
When culturing undifferentiated cells by a conventional monolayer culture method, it is difficult to induce differentiation of the entire cell group in a certain direction, that is, some cells are differentiated and some cells are undifferentiated. It remains as it is, it becomes a mixture of multiple types of cells having different functions, or the cells do not differentiate normally. In this regard, in the method of the present invention, an aggregate of cells is formed on the permeable membrane by applying centrifugal force or pressure to the undifferentiated cells, and by culturing the aggregates, the whole undifferentiated cell group is obtained. Efficiently and uniformly differentiate in a certain direction.
[0069]
Furthermore, by adding a component that induces cell differentiation to the aggregate culture environment, differentiation in a certain direction can be induced more efficiently.
[0070]
In addition, according to the method of the present invention, a cell tissue body having a three-dimensional structure having a homogeneous differentiation function can be obtained simply and efficiently.
[Brief description of the drawings]
FIG. 1 is a toluidine blue stained image of a chondrocyte culture obtained by inducing differentiation in one example of the differentiation inducing method of the present invention.
FIG. 2 is a toluidine blue-stained image of a chondrocyte culture obtained by inducing differentiation by a conventional monolayer culture method.
3 is a graph showing the amount of neutral fat accumulated in cells obtained in Example 2 and Comparative Example 2. FIG.
Claims (7)
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002336594A JP4186043B2 (en) | 2002-11-20 | 2002-11-20 | Cell differentiation inducing method and cell culture |
| AT03011907T ATE408666T1 (en) | 2002-05-28 | 2003-05-27 | METHOD OF CULTURE, STORAGE AND INDUCE DIFFERENTIATION OF CELLS AND DEVICE FOR USE IN SUCH METHOD, AND METHOD OF USE THEREOF. |
| DE60323561T DE60323561D1 (en) | 2002-05-28 | 2003-05-27 | A method of culture, storage and induction of differentiation of cells and apparatus for use in this method, and associated method of use. |
| EP03011907A EP1367119B1 (en) | 2002-05-28 | 2003-05-27 | Methods of culturing, storing, and inducing differentiation in cells, instrument for use in the methods and method of using the instrument. |
| US10/446,467 US20030224510A1 (en) | 2002-05-28 | 2003-05-28 | Methods of culturing, storing, and inducing differentiation in cells, instrument for use in the methods, method of using the instrument, and medicial biomaterial |
| US11/529,829 US20070020756A1 (en) | 2002-05-28 | 2006-09-29 | Methods of culturing, storing, and inducing differentiation in cells, instrument for use in the methods, method of using the instrument, and medical biomaterial |
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| JP2002336594A JP4186043B2 (en) | 2002-11-20 | 2002-11-20 | Cell differentiation inducing method and cell culture |
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