JP2008546410A - ヒト胚性幹細胞の懸濁培養法 - Google Patents
ヒト胚性幹細胞の懸濁培養法 Download PDFInfo
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Abstract
Description
本出願は、2005年6月22日に出願された米国特許出願第60/693266号の優先権を主張する。
再生医療は、様々な種類の始原細胞の分離、培養、利用に関連する最近の進歩によって恩恵を受けている。この発明の開示は、ヒト多能性幹細胞とその誘導体の商業的開発の更なる進歩を与えるものである。
本開示は、霊長類多能性幹細胞(hES細胞)の培養および増殖のための改良されたシステムを提供するものである。この発明に係る懸濁培養システムは、治療及び創薬における使用のために、早くかつ容量効率的に高品質の胚性幹細胞を生産させることができる。
霊長類の多能性幹(hES)細胞細胞を増殖させる従来の技術は、固体表面上での培養を含むものであった。即ち、繊維芽細胞フィーダー細胞法(米国特許第6,200,806号)、または細胞外マトリックス法(米国特許第6,800,480号)であった。フィーダーフリーの方法は、迅速な増幅を可能にするように最適化されるのでWO 03/020920、商業目的のhES細胞製造コストを大幅に減少させる。
始原型である「霊長類多能性幹細胞」(pPS細胞)は、受精後の任意の時期における前胚組織、胚組織、または胎児組織に由来する多能性細胞であり、正常な条件の下でいくつかの異なる細胞型の子孫を作ることが可能であるという特徴を有する。pPS細胞は、適当な宿主において奇形腫を形成する能力、または培養液において三つの胚葉全ての組織型のマーカーを持つ細胞に分化する能力のように、標準的な技術的に容認された試験に従って、三つの胚葉、即ち内胚葉、中胚葉、および外胚葉のそれぞれの誘導体である子孫を作ることができる。
分子遺伝学、並びに遺伝子工学の一般的方法は、Molecular Cloning: A Laboratory Manual, (Sambrook et al., Cold Spring Harbor);Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.);およびCurrent Protocols in Molecular Biology (F. M. Ausubel et al. eds., Wiley & Sons) の最新版に記載されている。細胞生物学、たんぱく質化学、および抗体技術は、Current Protocols in Protein Science (J. E. Colligan et al. eds., Wiley & Sons); Current Protocols in Cell Biology (J. S. Bonifacino et al., Wiley & Sons)、 およびCurrent Protocols in Immunology (J. E. Colligan et al. eds., Wiley & Sons)に見ることができる。この開示において引用される試薬、クローニングベクター、および遺伝子操作のためのキット類は、Biorad、Stratagene、Invitrogen、Clontech、およびSigma-Aldrich Co.等の民間の供給業者から入手可能である。
胚性幹細胞は、霊長類種のメンバーの胚盤胞から採取することができる(米国特許第5,843,780号; Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995)。ヒト胚性幹(hES)細胞は、Thomson et al. (米国特許第6,200,806号; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998)およびReubinoff et al. (Nature Biotech. 18:399, 2000)の技術に従って、初代マウス繊維芽細胞フィーダー細胞を用いてヒト胚盤胞細胞から調製することができる。hES細胞株はまた、ヒトフィーダー細胞上で(米国特許第6,642,048号)、または完全にフィーダーフリーの条件で(US 2002/0081724 A1)誘導されうる。hES細胞と同等の細胞型は、WO 01/51610 (Bresagen) において概説されたように、原始外胚葉様(EPL)細胞のような多能性派生物を含む。
最初にThomson (U.S. 5,843,780; U.S. 6,090,622)によって記載されたように、当初、この分野の科学者の多くは、分化を防ぐ目的でフィーダー細胞層の上でhES細胞を培養する方法を選んだ。
現在では、hES細胞は、固体の基質上で増殖させるよりもむしろ、懸濁培養によって増殖させることができることが発見されている。
・幹細胞因子(SCF, Steel factor)、c-kitを二量体化する他のリガンドまたは抗体、および同じシグナル伝達経路の他の活性化因子
・他のチロシンキナーゼ関連受容体に対するリガンド、例えば、血小板由来成長因子(PDGF), マクロファージコロニー刺激因子、Flt-3リガンド、および血管内皮成長因子(VEGF)の受容体
・サイクリックAMPレベルを上昇させる因子、例えば、フォルスコリン
・gp130を誘導する因子、例えば、LIFまたはオンコスタチン-M
・造血成長因子、例えば、トロンボポイチン(TPO)
・トランスフォーミング成長因子、例えば、TGFβ1
・他の成長因子、例えば上皮成長因子(EGF)
・ニューロトロフィン、例えばCNTF
この発明に従って培養されたヒトES細胞は、未分化幹細胞に特徴的な形態学的性質を持つ。二次元の標準的な顕微鏡像では、hES細胞は、画像の平面における高い核/細胞質比、よく目立つ核小体、およびよく識別できない細胞境界部を持つ密集したコロニー形成を有する。細胞株は、標準のGバンド法を用いて核型が調べられ(Oakland, CAのCytogenetics Labなどの日常業務の核型決定サービス業務を行う多くの臨床診断ラボが利用可能である)、公表されたヒト核型と比較される。細胞が正倍数体であり、顕著な変化をしていない全てのヒト染色体を持つ、「正常な核型」を持つ細胞を入手することが望ましい。
本発明は、多数の多能性細胞を商業的規模で生産する方法を提供するものである。該細胞は、未分化の状態で多数の研究および商業的目的で役に立つものであり、または、特定の細胞型へ分化させることに向けることができる。
肝細胞は、米国特許第6,458,589号およびPCT公報 WO 01/81549 (Geron Corporation) に記載されたように、ヒストンデアセチラーゼの阻害剤を用いてhES細胞から分化させることができる。未分化のhES細胞は、ヒストンデアセチラーゼの阻害剤の存在下で培養される。
神経細胞は、米国特許第6,833,269号; Carpentar et al., Exp. Neurol. 2001; 172(2):383-97; およびWO 03/000868 (Geron Corporation)に記載された方法に従ってhES細胞から生成されることができる。未分化hES細胞または胚様体細胞を、一つまたは複数のニューロトロフィン、および一つまたは複数の分裂促進物質を含有する培地で培養し、それにより少なくとも約60%の細胞がA2B5、ポリシアル酸化NCAM、またはネスチンを発現する細胞集団を生成し、かつこれは培養中に少なくとも20回の倍化を行うことができる。分裂促進物質の例は、EGF、塩基性FGF、PDGF、およびIGF-1である。ニューロトロフィンの例としては、NT-3およびBDNFがある。TGF-βスーパーファミリー拮抗剤の利用、またはcAMPとアスコルビン酸の組み合わせは、ドーパミン作動性ニューロンに特徴的なチロシンヒドロキシラーゼ陽性の神経細胞の比率を増加させるために使用することができる。増殖細胞は、さらに、分裂促進物質のない状態でニュートロフィンと共に培養することによって最終分化を行わせることができる。
心筋細胞または心筋細胞前駆体は、WO 03/006950に記載された方法によってhES細胞から生成させることができる。細胞は、ウシ胎児血清または血清代替物、および任意で5-アザシチジンなどのDNAのメチル化に影響する心臓の栄養因子とともに懸濁状態で培養される。また、心筋細胞の集団は、アクチビンAおよび骨形態形成たんぱく質4の組み合わせを用いて固体基質上で直接分化させることができる:かくして、収縮細胞を、密度遠心分離によって集団中の他の細胞から分離することができる。
膵島細胞は、アクチビンA、ヒストンデアセチラーゼ阻害剤(酪酸など)、分裂促進剤(bFGFなど)、およびTGF-βスーパーファミリー拮抗剤(ノギンなど)から選択されるいくつかの因子の組み合わせを含有する培地で培養することによってhES細胞の分化を開始させることにより、hES細胞から分化され得る。その後、該細胞をニコチンアミドと培養することによって成熟させることができ、細胞の少なくとも5%がPdx1、インスリン、グルカゴン、ソマトスタチン、および膵臓ポリペプチドを発現する細胞集団が得られる。WO 03/050249 (Geron Corp.)を参照されたい。
この発明に係る培養系の構成品は、売り物として提供され、販売され、またはそうでなければ、任意の目的での任意の企業による使用のために、製造場所から配布される。また、構成物は、次の二つまたはそれ以上の組み合わせの様々な有用な組み合わせで、一緒に販売または配布され得る。
・hES細胞を懸濁因子中で培養するための適当な培地
・培地に存在する、または添加されるべき細胞外マトリックス成分または増粘剤
・培地に存在する、または添加されるべきマイクロキャリア
・懸濁培養に合わせて改良された容器
・培養系において増殖している、または他の形態で貯蔵されるが培養系での使用が意図されたhES細胞それ自身
実施例1:迅速増殖培地における多能性幹細胞の成長
最初にマウス胚繊維芽細胞のフィーダー上で成長させ、それから米国特許第6,800,480号; Xu et al., Stem Cells 2005;23(3):315-23に詳しく述べられたようにMatrigel(商標登録)細胞外マトリックスと馴化培地を含むフィーダーフリー環境で20継代の間増殖させた、一系統のhES細胞を得た。
Matrigel(商標登録)上でMEF-CMにおいて培養されたhES細胞は、新鮮な(非馴化)血清非含有培地:グルタミン、非必須アミノ酸、およびβ-メルカプトエタノール+ Matrigel(商標登録)上の80 ng/mLヒト塩基性FGFで補足され、さらにヒトラミニンでコーティングされた表面に適合されたX-VIVO(商標)で継代された。あるいは、低温保存された細胞が、80 ng/mL hbFGFを含有する同じ培地に直接融解された。細胞は、コラゲナーゼIVを用いて5〜6日毎に継代された。
培養液の体積当りのhES細胞の収量を増やす目的で、細胞は懸濁培養され、次に、形態および三つ全ての胚葉を代表する分化した細胞になる能力が調べられた。
・容器:100 mLスピナーフラスコ
・接種(播種)密度:3.6 × 105 cells/mL
・培地量:スピナーフラスコ当り50 ml
・使用された培地:bFGF (8 ng/mL)を含有するmEF馴化培地
・攪拌速度:20 rpm (Bellco・キャリアー・マグネチック・スターラー)
・環境:37℃ CO2インキュベーター
・培地交換:隔日(細胞を沈下させ、上清を置換する)
H9 hES細胞は、これらの条件下で6日間スピナーフラスコ中で維持された。
・容器:100 mLスピナーフラスコ
・接種(播種)密度:3.5 × 105 細胞/mL
・培地量:スピナーフラスコ当り50 mL
・使用された培地: mEF-CM + bFGF (8 ng/mL)
・攪拌速度:20 rpm (前述)
・培地交換:三日に一度
この実験は、別のhES細胞株を用いて行われた。細胞は、いくつかの異なる培養条件下でスピナーフラスコの代わりにシェーカーフラスコを用いて培養され、培養は、2ヶ月以上継続された。
・容器:100 mLシェーカーフラスコ
・接種(播種)密度:5.0 × 105 細胞/mL
・培地量:シェーカーフラスコ当り15 ml
・攪拌速度:80 rpm (37℃ CO2インキュベーター中のLabline ローテータ/シェーカー)
・培地交換:最初は隔日、その後2〜3日毎
使用された培地および培養期間は次の通りである。
A: mEF-CM + bFGF (8 ng/mL)。98日間維持。
B: mEF-CM + bFGF (8 ng/mL) + ラミニン(最初に33 μg/mL、その後の残りの培養で約10μg/ml)。49日間維持。
C: X-VIVO(商標)10 + FGF (40 ng/mL) + Flt-3 (75 ng/mL)。11日間維持。
D: X-VIVO(商標) 10 + FGF (40 ng/mL) + Flt-3 (75 ng/mL) + ラミニン(最初に33 μg/mL、その後の残りの培養で約10μg/ml)。11日間維持。
培養期間中の細胞数は次の表に示される。
(表3)懸濁培養におけるhES細胞の増殖
次の実験では、懸濁培養において新鮮(非馴化)培地を用いる場合の代替えの添加物を評価する。
1) X-VIVO(商標) 10+ bFGF (80ng/mL)
2) X-VIVO(商標) 10+ bFGF (80ng/mL) + TGFβ1 (0.5 ng/mL)
3) X-VIVO(商標) 10+ bFGF (40ng/mL) + TGFβ1 (0.5 ng/mL)
4) X-VIVO(商標) 10+ bFGF (80ng/mL) + TGFβ1 (0.5 ng/mL) + 10μg/mLヒトラミニン
5) X-VIVO 10(商標)+ bFGF (80ng/mL) + TGFβ1 (0.5 ng/mL) + 50μg/mL ヒト血清アルブミン
Claims (20)
- hES細胞が実質的に未分化の状態にある、ヒト胚性幹(hES)細胞の懸濁培養。
- 細胞が少なくとも約40 ng/mLの濃度の繊維芽細胞成長因子を含有する培地で培養される、請求項1に記載の培養。
- 培地がトランスフォーミング成長因子ベータ(TGFβ)、幹細胞因子(SCF)、またはFlt3 リガンド(Flt3L)も含有する、請求項2に記載の培養。
- 細胞が一つまたは複数の、可溶性のまたは懸濁された細胞外マトリックス成分を含有する培地で培養される、前記請求項のいずれか一項に記載の培養。
- 細胞外マトリックス成分がヒトラミニンおよび/またはヒトフィブロネクチンを含む、請求項4に記載の培養。
- 細胞が固体微粒子と共に懸濁培養される、前記請求項のいずれか一項に記載の培養。
- 微粒子が、一つまたは複数の、可溶性のまたは懸濁された細胞外マトリックス成分でコーティングされる、請求項7に記載の培養。
- hES細胞を培養する方法であって、以下の工程を含む方法:
a) 該細胞を栄養培地に懸濁する工程;
b) 培養している間、該細胞を懸濁状態に保つ工程;
c) 培地を定期的に交換する工程;
d) 必要に応じて培養液を時々分割し、細胞密度を減少させる工程;および最後に、
e) 培養液から細胞を収集する工程。 - 細胞が少なくとも約40 ng/mLの濃度の繊維芽細胞成長因子を含有する培地で培養される、請求項8に記載の方法。
- 培地がトランスフォーミング成長因子ベータ(TGFβ)、幹細胞因子(SCF)、またはFlt3リガンド (Flt3L)も含有する、請求項9に記載の方法。
- 細胞が一つまたは複数の、可溶性のまたは懸濁された細胞外マトリックス成分を含有する培地で培養される、請求項8〜10のいずれか一項に記載の方法。
- 細胞外マトリックス成分がヒトラミニンおよび/またはヒトフィブロネクチンを含む、請求項11に記載の方法。
- 細胞が固体微粒子と共に懸濁培養される、請求項8〜12のいずれか一項に記載の方法。
- 微粒子が、一つまたは複数の、可溶性のまたは懸濁された細胞外マトリックス成分でコーティングされる、請求項13に記載の方法。
- 細胞が少なくとも2ヶ月間懸濁培養される、請求項8〜14のいずれか一項に記載の方法。
- 細胞が、懸濁培養される間に少なくとも3倍増殖される、請求項8〜15のいずれか一項方法。
- 実質的に未分化の細胞集団が維持されるように、収集した細胞を固体表面にプレーティングして、該細胞の培養を継続する工程を更に含む、請求項8〜16のいずれか一項に記載の方法。
- 収集された細胞を分化させる工程を更に含む、請求項8〜17のいずれか一項に記載の方法。
- 少なくとも約40 ng/mLの繊維芽細胞成長因子を含有する栄養培地を含む、hES細胞を懸濁培養するためのシステムまたはキット。
- 培地に添加するための細胞外マトリックス成分または微粒子も含む、請求項19に記載のシステムまたはキット。
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JP2012507281A (ja) * | 2008-11-04 | 2012-03-29 | バイアサイト インク | 幹細胞集合体懸濁液組成物、その分化方法 |
US10876094B2 (en) | 2009-11-12 | 2020-12-29 | Technion Research & Development Foundation Limited | Culture media, cell cultures and methods of culturing pluripotent stem cells in an undifferentiated state |
JP2022088599A (ja) * | 2009-11-12 | 2022-06-14 | テクニオン リサーチ アンド ディベロップメント ファウンデーション リミテッド | 多能性幹細胞を未分化状態で培養する培地、細胞培養および方法 |
JP7383069B2 (ja) | 2009-11-12 | 2023-11-17 | テクニオン リサーチ アンド ディベロップメント ファウンデーション リミテッド | 多能性幹細胞を未分化状態で培養する培地、細胞培養および方法 |
JP2014036660A (ja) * | 2013-10-09 | 2014-02-27 | Viacyte Inc | 幹細胞集合体懸濁液組成物、その分化方法 |
WO2016088243A1 (ja) * | 2014-12-05 | 2016-06-09 | 株式会社ニコン | 判定装置、観察システム、観察方法、そのプログラム、細胞の製造方法、および細胞 |
WO2021033722A1 (ja) * | 2019-08-20 | 2021-02-25 | 昭和電工マテリアルズ株式会社 | 馴化細胞作製方法、馴化細胞管理方法、馴化細胞管理装置、及びコンピュータプログラム |
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