JP2021090463A - 間葉系幹細胞の細胞培養法 - Google Patents
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Abstract
Description
基礎培地と;
血小板由来成長因子(PDGF)と;
線維芽細胞増殖因子2(FGF2)と;
を含有し、ここでの前記培地中のFGF2のレベルは、約6ng/mL未満である。
血小板由来成長因子(PDGF)と;
線維芽細胞増殖因子2(FGF2)と;
を含有し、
ここでの前記FGF2は、約6ng/mL未満のレベルで幹細胞培養培地に添加されるのに十分な量で組成物中に存在する。別の例において、FGF2は、約5pg/mL〜40pg/mLのレベルで幹細胞培養培地に添加されるのに十分な量で、前記組成物中に存在する。別の例において、FGF2は、約20pg/mLのレベルで幹細胞培養培地に添加されるのに十分な量で、前記組成物中に存在する。
特に別途定義しない限り、本明細書で使用する全ての技術的及び科学的用語は、当該技術(例えば、細胞培養、分子生物学、幹細胞培養、タンパク質化学、及び生化学)における通常の専門家が一般的に理解するのと同じ意味を有するものと解される。
本明細書中で使用するとき、「間葉系統前駆細胞または幹細胞」の用語は、未分化の多能性細胞を意味し、これは多分化能を維持しながら自己再生する能力を有し、且つ間葉起源の多くの細胞型、例えば骨芽細胞、軟骨細胞、脂肪細胞、間質細胞、線維芽細胞及び腱、または非中胚葉起源、例えば肝細胞、神経細胞及び上皮細胞の何れかのへと分化する能力を有する。疑義を回避するために、「間葉系統前駆細胞」は、骨、軟骨、筋肉及び脂肪細胞のような間葉細胞、及び繊維性結合組織に分化できる細胞を言う。
本開示の一態様は、インビトロ細胞培養において、幹細胞増殖を促進する方法に関する。本開示の関連において、「促進する」または「促進すること」の用語は、細胞増殖における増大または加速を規定するために使用される。
幹細胞は非対称に分裂して2つの異なる娘細胞、すなわち、元の幹細胞のコピー1つと、最終的な非幹細胞へと分化するようにプログラムされた第2の娘細胞を生じる。例えば、多能性幹細胞は、1コピーの元の幹細胞、並びに間葉系細胞型へと分化するようにプログラムされた間葉系統前駆細胞へと分裂することができる。
本開示は、MSC増殖を促進する成長因子を含んだ、ウシ胎仔血清フリーの幹細胞培地を提供する。一実施形態において、本開示は、ウシ胎仔血清フリーの幹細胞培養培地に関し、該細胞培養培地は、
基礎培地と;
血小板由来成長因子(PDGF)と;
線維芽細胞増殖因子2(FGF2)と
を含有する。
アスコルビン酸は、培養中の様々な種類の細胞の増殖及び分化のために必須のサプリメントである。特定のアスコルビン酸誘導体は、溶液中、特に中性のpH及び37℃の通常の細胞培養条件下の溶液中において安定ではないので、今は「短時間作用性」であると理解されている。これらの短時間作用性誘導体は、急速にシュウ酸またはトレオニン酸へと酸化される。37℃の培養培地(pH7)において、酸化は、これら短時間作用性アスコルビン酸誘導体のレベルを24時間で約80〜90%低下させる。従って、短時間作用性アスコルビン酸誘導体は、様々な細胞型の従来の細胞培養において、より安定な「長時間作用性」アスコルビン酸誘導体に置き換えられている。
従来、幹細胞は、少なくとも約10〜15%v/vの血清、一般的にはウシ胎仔血清(FBS)(子ウシ胎仔血清(FCS)としても知られている)を補充した培地を用いて、細胞培養物中に維持されている。本開示の細胞培養培地は、ウシ胎仔血清フリーの細胞培養培地である。従って、一実施形態では、細胞培養培地に非胎仔血清を補充する。例えば、培地に新生仔または成体血清を補充してよい
本発明者等は、PDGF及びFGF2が、インビトロでのウシ胎仔血清フリーの細胞培養において幹細胞増殖を相乗的に促進することを見出した。
本開示の細胞培養培地は、細胞培養の前に、基礎培地と成長因子が一緒に混合されている完全培地として提供することができる。或いは、細胞培養培地成分を別々に提供し、細胞培養の前または最中に適切な基礎培地と混合することができる。従って、一実施形態において、本開示は、幹細胞培養培地に添加するための血清代用組成物を提供し、該組成物は、
血小板由来成長因子(PDGF)と;
線維芽細胞増殖因子2(FGF2)と;
を含有し、ここで前記組成物が幹細胞培養培地に添加され、且つ幹細胞集団が該細胞培養培地中で培養される場合、培養培地中のFGF2のレベルは約6ng/mL未満である。他の例において、前記組成物が幹細胞培養培地に添加され、幹細胞集団が該細胞培養培地中で培養される場合、FGF2レベルは約5ng/mL未満、約4ng/mL未満、約3ng/mL未満、約2ng/mL未満、約1ng/mL未満である。他の例において、FGF2レベルは、約0.9ng/mL未満、約0.8ng/mL未満、約0.7ng/mL未満、約0.6ng/mL未満、約0.5ng/mL未満、約0.4ng/mL未満、約0.3ng/mL未満、約0.2ng/mL未満である。
ヒト血小板溶解物中における間葉系統前駆細胞(MPC)の連続的な増殖を評価するために、細胞増殖を測定する前に、ヒト血小板溶解物中で細胞を7日間培養した。血小板溶解物は、2つの別々のサプライヤ(サプライヤA及びサプライヤB)から得たものである。血小板溶解産物のパーセンテージは0〜10%の範囲であった。7日目のMPC増殖を図1に示す。細胞増殖は、サプライヤBから得た10%ヒト血小板溶解物中で細胞を増殖させた場合に最も高かいことが注目された。
細胞増殖に対するhPL中の成長因子の影響を評価するために、細胞集団を成長因子EGF、FGF2、VEGF、PDGF−AA及びPDGF−BBの抗体アンタゴニストに暴露させた。
何れの他の成長因子がPDGF−BBによるMPC増殖に寄与しているかを評価するために、MPC細胞を、図3に示す抗体の様々な組み合わせと共に培養した。
出発基礎細胞培養培地は、アルファ改変イーグル最小必須培地(MEM)またはStemSpan(商標)を含んでいた。
・PDGF−BB(3.2ng/mL)、EGF(0.08ng/mL)及びFGF2(0.002ng/mL)
・PDGF−BB(9.6ng/mL)、EGF(0.24ng/mL)及びFGF2(0.006ng/mL)
・PDGF−BB(16ng/mL)、EGF(0.4ng/mL)及びFGF2(0.01ng/mL)
・PDGF−BB(32ng/mL)、EGF(0.8ng/mL)及びFGF2(0.02ng/mL)
凍結保存されたヒトMPCを解凍し、組換えヒト成長因子を補充したゼノフリー培地、または成長因子を含まないゼノフリー培地中において、96ウェルプレート上に1,000細胞/ウェルで播種した。
・PDGF−BB(P)−10ng/mL
・EGF(E)−5ng/mL
・FGF2(F)−1ng/mL
・PDGF、EGF、FGF2(PEF)
3つの異なるドナーからのMPCを、標準培地(アルファMEM+10%FBS)、またはアルファMEM+3%ヒトAB血清+PDGF−BB、EGF及びFGF2を含むウシ胎仔血清フリーの培地中において連続的に増殖させた。サイトカインレベルを、細胞増殖後の細胞培養培地中で測定した(図11)。
Claims (16)
- 幹細胞増殖をインビトロで促進する方法であって:
血小板由来成長因子(PDGF)及び線維芽細胞増殖因子2(FGF2)を含むウシ胎仔血清フリーの細胞培養培地中において、間葉系統幹細胞の集団を培養することを含み、ここでのFGF2のレベルは約6ng/mL未満である前記方法。 - 幹細胞増殖をインビトロで促進するためのウシ胎仔血清フリーの細胞培養培地であって:
基礎培地と;
血小板由来成長因子(PDGF)と;
線維芽細胞増殖因子2(FGF2)と;
を含有し、
前記培地中のFGF2のレベルが約6ng/mL未満である前記細胞培養培地。 - 前記FGF2のレベルが少なくとも約2pg/mL〜40pg/mLである、請求項1に記載の方法または請求項2に記載の培養培地。
- 前記FGF2のレベルが約20pg/mLである、請求項1に記載の方法または請求項2に記載の培養培地。
- 前記PDGFがPDGF−BBである、請求項1〜4の何れか1項に記載の方法または培養培地。
- 前記PDGF−BBのレベルが少なくとも約3.0ng/mL〜約120ng/mLである、請求項5に記載の方法または培養培地。
- 前記PDGF−BBのレベルが、約9.0ng/mL〜約60ng/mLである、請求項5に記載の方法または培養培地。
- 前記PDGF−BBのレベルが少なくとも30ng/mLである、請求項5に記載の方法または培養培地。
- 前記細胞培養培地が更に上皮成長因子(EGF)を含む、請求項1〜8の何れか1項に記載の方法または培養培地。
- 前記EGFのレベルが、約0.08ng〜約7ng/mLである、請求項9に記載の方法または培養培地。
- 前記EGFのレベルが、約0.4ng/mL〜約1.6ng/mLである、請求項9に記載の方法または培養培地。
- 前記EGFのレベルが、少なくとも約0.8ng/mLである、請求項9に記載の方法または培養培地
- 前記細胞培養培地が、アルファ最小必須培地またはウシ胎仔血清フリーの増殖培地を含有する、請求項1〜12の何れか1項に記載の方法または培養培地。
- 前記細胞培養培地が前記幹細胞を未分化状態に維持する、請求項1〜13の何れか1項に記載の方法または培養培地。
- 前記幹細胞が間葉系統前駆細胞である、請求項1〜14の何れか1項に記載の方法または培養培地。
- 前記幹細胞が間葉系幹細胞である、請求項1〜15の何れか1項に記載の方法。
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EP2334785B2 (en) | 2008-09-16 | 2019-08-14 | Mayo Foundation For Medical Education And Research | Compositions containing platelet contents |
US11891620B2 (en) | 2014-05-16 | 2024-02-06 | Mayo Foundation For Medical Education And Research | Cell culture media compositions for primary cells |
CN108251358B (zh) * | 2017-12-15 | 2021-10-26 | 广东药科大学 | 一种相同供体来源人间充质干细胞的多批次原代分离方法 |
US20210115395A1 (en) * | 2018-03-28 | 2021-04-22 | Pluristem Ltd. | Methods for expanding adherent stromal cells and cells obtained thereby |
CN108624554B (zh) * | 2018-05-14 | 2021-09-14 | 山东大学 | 一种非基因修饰光操控双向调节人脐带间充质干细胞增殖的方法 |
US11364323B2 (en) | 2018-09-17 | 2022-06-21 | Rejuvablast LLC | Combination grafts for tissue repair or regeneration applications |
JPWO2020067443A1 (ja) * | 2018-09-27 | 2021-08-30 | テルモ株式会社 | 体細胞のシート化方法 |
EP4165166A2 (en) * | 2020-06-11 | 2023-04-19 | Mesoblast International Sàrl | 3d culture of mesenchymal lineage precursor or stem cells |
WO2022192446A1 (en) * | 2021-03-10 | 2022-09-15 | Terasaki Institute For Biomedical Innovation | Production of heme for cell-based meat products |
WO2024121821A1 (en) | 2022-12-09 | 2024-06-13 | Mesoblast International Sarl | Conditioned media and use of the same |
WO2024121819A1 (en) | 2022-12-09 | 2024-06-13 | Mesoblast International Sarl | Method of treating inflammation with cellular compositions |
WO2024121820A1 (en) | 2022-12-09 | 2024-06-13 | Mesoblast International Sarl | Pre-licensed composition and cell culture methods |
WO2024121818A1 (en) | 2022-12-09 | 2024-06-13 | Mesoblast International Sarl | Method of treating heart failure in subjects with persistent inflammation |
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CN113957043A (zh) | 2022-01-21 |
JP2024001179A (ja) | 2024-01-09 |
WO2016139340A1 (en) | 2016-09-09 |
BR112017018885A2 (pt) | 2018-04-17 |
EP3265554B1 (en) | 2021-11-17 |
JP7410899B2 (ja) | 2024-01-10 |
SG11201706886PA (en) | 2017-09-28 |
CA2977578A1 (en) | 2016-09-09 |
JP6885870B2 (ja) | 2021-06-16 |
AU2021254538B2 (en) | 2024-01-18 |
EP4008775A1 (en) | 2022-06-08 |
US20180037867A1 (en) | 2018-02-08 |
KR102635212B1 (ko) | 2024-02-13 |
JP2018506986A (ja) | 2018-03-15 |
KR20170120627A (ko) | 2017-10-31 |
EP3265554A1 (en) | 2018-01-10 |
US20240191200A1 (en) | 2024-06-13 |
CN107567494A (zh) | 2018-01-09 |
US11685899B2 (en) | 2023-06-27 |
AU2016227607A1 (en) | 2017-09-07 |
AU2021254538A1 (en) | 2021-11-18 |
US20200040303A1 (en) | 2020-02-06 |
AU2016227607B2 (en) | 2021-09-30 |
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