JP7114468B2 - 支持されてインビトロで発達した組織培養物と培養法 - Google Patents
支持されてインビトロで発達した組織培養物と培養法 Download PDFInfo
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Description
a)横長の配置または長め配置にて配置された、および/または繊維性支持体の上に配置された、複数の多能性細胞または非ヒト全能性細胞を提供する工程と、
b)前記細胞を前記配置で増殖させて分化させる工程であって、ここで前記細胞が細胞間接着を形成して互いに接着する工程、を含み;そのような方法によって得ることができる、複能性細胞の細長い多細胞凝集塊または繊維支持多細胞凝集塊が提供される。
o)複能性細胞の細長い多細胞凝集塊または繊維支持多細胞凝集塊を提供し、
p)前記細長い多細胞凝集塊または繊維支持多細胞凝集塊を三次元マトリックスの中で培養して前記細胞を分化させることによって前記細胞を増殖させ、
q)工程p)で増殖した細胞を懸濁培養物の中で培養すること、を含んでいる。
u)複能性細胞の多細胞凝集塊を提供し、
v)その多細胞凝集塊を三次元マトリックス(ゲルが好ましい)の中で培養して前記細胞を分化させることによって前記細胞を増殖させ、
w)工程v)で増殖した細胞を懸濁培養物の中で培養し、
r)三次元マトリックスが溶解した材料を前記懸濁培養物に添加し、それによって、前記溶解した材料が前記培養物に接着してマトリックスを形成すること、を含む方法も提供される。
図1aに示したように、本発明は、改良されたオルガノイドを形成する方法に関する。この方法は少なくとも2つの部分の方法に分割することができ、それらの部分は、すでに独立に有効な結果を提供する。すなわち第1の工程群により、その後の任意の培養法において有利な発生能力をすでに有する、細胞の細長い凝集塊または繊維支持凝集塊が得られる。細胞のこの細長い凝集塊または繊維支持凝集塊は、いくつかの定義によれば胚様体であり、本発明の1つの側面も形成する。図1aに示した第2の工程群では、細胞の凝集塊をさらに発達させて、改善された特徴を持つ人工組織培養物にする。この方法と得られる人工組織培養物(オルガノイドとも呼ばれる)は、本発明のさらに別の、だが関連した側面を形成する。
a)横長の配置または長めの配置において配置された複数の多能性細胞または非ヒト全能性細胞を提供する工程と、
b)前記細胞を前記配置で増殖させて分化させる工程であって、そこで前記細胞が細胞間接着(例えば結合(bonds)または接合(junctions))を形成して互いに接着する、工程、を含んでいる。
o)好ましくは上に規定した複能性細胞の細長い多細胞凝集塊または繊維支持多細胞凝集塊を提供し、
p)その細長い多細胞凝集塊または繊維支持多細胞凝集塊を三次元マトリックス(ゲルが好ましい)の中で培養して前記細胞を分化させることによってそれら細胞を増殖させ、
q)工程p)で増殖した細胞を懸濁培養物の中で培養すること、を含む方法も提供される。
u)好ましくは上に規定した複能性細胞の多細胞凝集塊を提供し、
v)その多細胞凝集塊を三次元マトリックス(ゲルが好ましい)の中で培養して前記細胞を分化させることによってそれら細胞を増殖させ、
w)工程v)で増殖した細胞を懸濁培養物の中で培養し、
r)三次元マトリックスが溶解した材料を前記懸濁培養物に添加することにより、その溶解した材料がその培養物に接着してマトリックスを形成することを含み、
好ましくは三次元マトリックスが溶解した前記材料が、溶解した細胞外マトリックスである方法も提供される。
a)横長の配置または長めの配置にて配置された複数の多能性細胞または非ヒト全能性細胞を提供する工程と、
b)前記細胞を前記配置で増殖させて分化させる工程であって、ここで前記細胞が細胞間接着を形成して互いに接着する、工程、を含む方法。
a)繊維性支持体の上に配置された複数の多能性細胞または非ヒト全能性細胞を提供する工程と、
b)前記細胞を前記配置で増殖させて分化させる工程であって、ここで前記細胞が細胞間接着を形成して互いに接着する、工程、を含む方法。
o)複能性細胞の細長い多細胞凝集塊または繊維で支持された多細胞凝集塊、好ましくは実施態様12~17のいずれか1項に規定されている細胞凝集塊を提供する工程、
p)前記細長い多細胞凝集塊または繊維支持多細胞凝集塊を三次元マトリックス、好ましくはゲルの中で培養する工程であって、ここで前記細胞を分化させることによって、前記細胞を増殖させる、工程、
q)工程p)で増殖した細胞を懸濁培養物の中で培養する工程、を含む方法。
を含む、実施態様18に記載の方法。
u)複能性細胞の多細胞凝集塊、好ましくは実施態様12~17のいずれか1項に規定されている多細胞凝集塊を提供し、
v)前記多細胞凝集塊を三次元マトリックス、好ましくはゲルの中で培養し、ここで前記細胞を分化させることによって前記細胞を増殖させ、
w)工程v)で増殖した細胞を懸濁培養物の中で培養し、
r)三次元マトリックスが溶解した材料を前記懸濁培養物に添加し、それによって、前記溶解した材料が前記培養物に接着してマトリックスを形成し、
好ましくは三次元マトリックスが溶解した前記材料が、溶解した細胞外マトリックスである、方法。
を含む方法。
ビタミンC;ビタミンA、2-メルカプトエタノール;bFGF;ROCK阻害剤;インスリン;GSK3β阻害剤;Wntアクチベータ(CHIR 99021が好ましい);抗菌剤(ペニシリンおよび/またはストレプトマイシンが好ましい);SMAD阻害剤(ドルソモルフィンおよび/またはSB-431542が好ましい);レチノイド(レチノイン酸が好ましい)から選択される少なくとも1つの化合物、またはこれらの任意の組み合わせ
を含む(GSK3β阻害剤かつWntアクチベータ(CHIR 99021が好ましい)を含むことが好ましい)キット。
で計算した。
Claims (32)
- ニューロン分化細胞を有する神経系譜の、細長いまたは繊維で支持された多細胞凝集塊を作製する方法であって、
a)(i)支持体(ここで前記支持体が20μm~20mmの長さ、および1μm~60μmの直径を有し、ここで前記支持体がバイオポリマーではない生体適合性ポリマーであるか、または前記支持体がタンパク質である)に接着した配置にて配置された、あるいは、(ii)繊維構造支持体(ここで前記繊維構造支持体が20μm~20mmの長さ、および1μm~60μmの直径を有し、ここで前記繊維構造支持体がバイオポリマーではない生体適合性ポリマーであるか、または前記繊維構造支持体がタンパク質である)上に配置された、複数の多能性細胞(pluripotent cells)または非ヒト全能性細胞(non-human totipotent cells)を提供する工程であって、ここで前記長さは、前記支持体の端部と端部の間の湾曲に沿った最も長い距離に従って決まるものであり、前記直径は、前記支持体の最も広い幅である、工程と、
b)前記細胞を前記配置で増殖させて分化させる工程であって、ここで前記細胞が細胞間結合を形成して互いに接着する、工程、
を含み、ここで前記細胞は、前記細胞をニューロン増殖因子またはニューロン分化因子と接触させることによって、刺激して分化させる、方法。 - 前記配置は、扁長寸法と垂直寸法のアスペクト比が少なくとも2:1である、請求項1に記載の方法。
- 前記支持体が、非多孔性である、または前記支持体の体積の5%(v/v)未満の空孔率を有する;または、前記支持体が、20μm~20mmの長さ、および/または1μm~60μmの直径を有する、請求項1または2に記載の方法。
- 前記支持体が、ポリマーマイクロフィラメントである、および/または生体適合性であるが生物活性ではない、請求項1~3のいずれか1項に記載の方法。
- 前記支持体がポリエチレングリコール鎖を含む、請求項1~4のいずれか1項に記載の方法。
- 前記支持体が、ポリラクチド、ポリグリコリド、もしくはこれらの組み合わせを含むか、または、ポリラクチド、ポリグリコリド、もしくはこれらの組み合わせからなる、請求項5に記載の方法。
- 前記支持体は、工程b)の後に溶解されるか、または生体吸収される、請求項1~6のいずれか1項に記載の方法。
- 前記工程a)において、2個~50万個の細胞が前記配置にて配置される、請求項1~7のいずれか1項に記載の方法。
- 最も離れた細胞が少なくとも1μm離れている、請求項1~8のいずれか1項に記載の方法。
- 前記支持体が、1~50個の非多孔性のマイクロフィラメントを含む、請求項1~9のいずれか1項に記載の方法。
- ニューロン分化細胞を有する神経系譜の、細長い多細胞凝集塊または繊維で支持された多細胞凝集塊であって、ここで細胞は、繊維構造支持体上に、扁長寸法と垂直寸法のアスペクト比が少なくとも2:1である配置にて配置されており、ここで前記繊維構造支持体がバイオポリマーではない生体適合性ポリマーであるか、または前記繊維構造支持体がタンパク質であり、そしてここで前記繊維構造支持体が20μm~20mmの長さ、および1μm~60μmの直径を有し、ここで前記凝集塊は、異なる分化段階の細胞を含有し、前記細胞塊は極性細胞および前記凝集塊の中心に対して一様な配向を有する前記極性細胞を含有するものであり、ここで前記長さは、前記支持体の端部と端部の間の湾曲に沿った最も長い距離に従って決まるものであり、前記直径は、前記支持体の最も広い幅である、多細胞凝集塊。
- 前記極性細胞は、前記凝集塊の細胞の少なくとも50%を構成する、請求項11に記載の凝集塊。
- 8000~1億個の細胞を含み、および/または50μm~40mmのサイズを有する、請求項11または12に記載の凝集塊。
- 前記支持体が、1~50個の非多孔性のマイクロフィラメントを含む、請求項11~13のいずれか1項に記載の凝集塊。
- 人工ニューロン組織培養物を作製する方法であって、
o)請求項11に規定されている、神経系譜の、細長い多細胞凝集塊または繊維で支持された多細胞凝集塊を提供する工程、
p)前記細長い多細胞凝集塊または繊維支持多細胞凝集塊を、三次元マトリックス中で培養する工程であって、ここで前記細胞を分化させることによって、前記細胞を増殖させる、工程、
q)工程p)で増殖した細胞を懸濁培養物の中で培養する工程
を含む、方法。 - さらに、
r)三次元マトリックスが溶解した材料を前記懸濁培養物に添加し、それによって前記溶解した材料が前記培養物に接着してマトリックスを形成する工程、
を含む、請求項15に記載の方法。 - 三次元マトリックスが溶解した前記材料が、溶解した細胞外マトリックスである、請求項16に記載の方法。
- 人工ニューロン組織培養物を作製する方法であって、
u)請求項11に規定されている、神経系譜の多細胞凝集塊を提供する工程、
v)前記多細胞凝集塊を、三次元マトリックス中で培養する工程であって、ここで前記細胞を分化させることによって前記細胞を増殖させる、工程、
w)工程v)で増殖した細胞を懸濁培養物の中で培養する工程、
r)三次元マトリックスの溶解した材料を前記懸濁培養物に添加し、それによって、前記溶解した材料が前記培養物に接着してマトリックスを形成する工程であって、ここで三次元マトリックスが溶解した前記材料が、溶解した細胞外マトリックスである、工程
を含む方法。 - 前記三次元マトリックスがゲルである、請求項15~18のいずれか1項に記載の方法。
- 請求項1~10および15~19のいずれか1項に記載の方法であって、さらに、
請求項1~10および15~19のいずれか1項に記載の方法を実施中の任意の段階で、さらに、
i)請求項1~10および15~19のいずれか1項に記載の方法の細胞における対象の遺伝子の発現を低下または増加させるか、または
ii)候補薬を、請求項1~10および15~19のいずれか1項に記載の方法の細胞に投与すること
を含み、それによって前記遺伝子または候補薬の発生における組織の効果を調査することを特徴とする、方法。 - すべての前記段階で、前記候補薬を前記細胞に投与すること、を含む、請求項20に記載の方法。
- 請求項15~19のいずれか1項に記載の方法によって得ることができる人工ニューロン組織培養物。
- 放射状に組織化された皮質板を含む人工ニューロン組織培養物であって、ここで前記組織培養物が、細胞の凝集塊からインビトロで増殖したものである、および/またはインビボで発達した脳もしくはその組織サンプルの培養物ではない、人工ニューロン組織培養物であって、
ラミニンを含む基底膜;神経上皮の基底面を覆う基底膜;移動するニューロンの外側の基底膜;あるいは、発現マーカーCtip2、Map2、DCXまたはその任意の組み合わせ(特にCtip2、Map2およびDCX)を含む放射状に組織化された皮質板、
を含む、人工ニューロン組織培養物。 - さらに、Map2または放射状グリアを含む、請求項23に記載の組織培養物。
- 放射状グリアとニューロンの直線状ユニットを含む、請求項23~24のいずれか1項に記載の組織培養物。
- 発生効果、特に先天性異常効果に関する候補薬をテストするまたはスクリーニングする方法であって、
候補薬を、請求項23に記載の人工培養物に投与すること、または、請求項15~19のいずれか1項に記載の方法を実施中に投与すること、および
前記培養物の前記細胞の対象の活性を決定し、前記活性を、前記候補薬を投与していない培養物の細胞の活性と比較することであって、ここで活性の違いが発生効果を示すこと、
を含む、方法。 - 請求項15~19および26のいずれか1項に記載の方法におけるキットの使用であって、前記キットは、
i)棒形構造体または格子形構造体または繊維状構造体の固体支持体であって、ここで前記支持体は20μm~20mmの長さ、および1μm~60μmの直径を有するものであって、ここで前記長さは、前記支持体の端部と端部の間の湾曲に沿った最も長い距離に従って決まるものであり、前記直径は、前記支持体の最も広い幅である、支持体と、
ii)三次元マトリックス、および/または、コラーゲン、ラミニン、エンタクチン、およびヘパリン-硫酸化プロテオグリカン、もしくはこれらの任意の組み合わせから選択される細胞外マトリックスまたはその任意の成分
を含む、使用。 - 前記キットは、Engelbreth-Holm-Swarm腫瘍由来の細胞外マトリックス、またはマトリゲルを含む、請求項27に記載の使用。
- 前記キットは、ビタミンC;ビタミンA、2-メルカプトエタノール;bFGF;ROCK阻害剤;インスリン;GSK3β阻害剤;Wntアクチベータ、好ましくはCHIR 99021;抗菌剤;SMAD阻害剤;レチノイド;またはこれらの任意の組み合わせ、をさらに含む、請求項27または28に記載の使用。
- 前記三次元マトリックスがゲルである、請求項27~29のいずれか1項に記載の使用。
- 請求項15~19および26のいずれか1項に記載の方法におけるキットの使用であって、前記キットは、
棒形構造または格子形構造または繊維構造支持体であって、前記支持体が20μm~20mmの長さ、および1μm~60μmの直径を有する支持体であって、ここで前記長さは、前記支持体の端部と端部の間の湾曲に沿った最も長い距離に従って決まるものであり、前記直径は、前記支持体の最も広い幅である、支持体と、
ビタミンC;ビタミンA、2-メルカプトエタノール;bFGF;ROCK阻害剤;インスリン;GSK3β阻害剤;Wntアクチベータ;抗菌剤;SMAD阻害剤;レチノイドから選択される少なくとも1つの化合物、またはこれらの任意の組み合わせ
を含む、使用。 - 前記固体支持体が、非多孔性である、および/または請求項3~6のいずれか1項に規定されたものである、請求項27~30のいずれか1項に記載の使用。
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