JP2016533745A - 多能性幹細胞及び前駆細胞を生産するためのプロセス - Google Patents
多能性幹細胞及び前駆細胞を生産するためのプロセス Download PDFInfo
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Abstract
Description
幹細胞群は、ヒトの体の特定の解剖学的な位置(即ち、天然ニッチ)に存在していることが知られている。天然ニッチは、適切な恒常性及び組織修復の関与並びにこれらの細胞の分裂を可能にするのに必要なこれらの維持管理及び細胞相互作用を保証するものである。従って、幹細胞ニッチは、細胞の未分化状態の維持管理に関する狭い範囲での細胞間シグナルを提供する微小環境において、その細胞外マトリクスと調和的に相互作用して存在している数種類の細胞を含む任意の組織の機能的ユニットである。
本発明は、成人幹細胞、特に多能性間葉系/間質幹細胞及び血管周囲前駆体(例えば周皮細胞)を生産するための革新的なプロセスに関する。
A- 1又は複数のNSCNの取得ステップ;
B- 1又は複数のNSCNの前調製ステップ;
C- 1又は複数のNSCN組織断片の調製ステップ;
D- 基礎培地においてNSCN断片を培養することによるSC増殖の促進ステップ;
E- NSCN断片からのSCの分離ステップ;
F- 任意に、Eにおける分離されたNSCNをステップDに戻すステップ。
臍帯の処理及び細胞培養
この実施例の記述は、同様に実行される無数の実施形態のうちの平均な数を表す。
最新の酵素法と本発明の方法との比較
臍帯を単離して、3つの等しいパーツ(ほぼ5cm)に分けた。第一パーツは、実施例1に従って処理して、生育培地に直接移した。第二及び第三パーツを洗浄して、実施例1にて説明したように断片化処理し、そして、コラゲナーゼ(0.1%のコラゲナーゼ、2時間)及びTrypLE(登録商標)(30分間)でそれぞれ処理した。図1の通り、細胞集団間の相違が、培養の72時間後(A、B及びC)及び5日後(A1、B1及びC1)に、これらの3つの方法によって観察された。A及びA1において、コロニーを形成し始めている接着された細胞の量だけでなく、より明確に紡錘状である、細胞形態の相違を観察することができる。これらの細胞の増殖は、等しい数の細胞(25cm2に103)を平板播種することによって評価した。本発明にかかる細胞が5日目に106細胞量を有する90%コンフルエンスに達して凍結させた一方で、酵素法によって得られた細胞は70%コンフルエンスにしか達しなかったことが観察された(下記の表1を参照)。継代の数は、断片の移送の間、計数しなかった。それゆえに、それぞれの移送は、継代数0の細胞を生産する。従って、断片の複数回の移送を考慮すると、それらの治療的な使用上の限界継代として計算される低継代(最大P5又は継代5)にある細胞数は事実上無限である。
表1 図1の代替酵素法に対する本発明の態様の比較。
インビトロで培養された、本発明のプロセスによって得られた細胞のフローサイトメトリーによる特性評価。
フローサイトメトリーによる分析のために、細胞表面分子及びそれらの各コントロールアイソタイプに対する抗体(例えば、ヒト抗CD45モノクローナル(シグマ社、米国)CD90(BD-Pharmigen社(米国))及びCD105、CD73(Serotec社(英国)))を使用した。100万の細胞を、氷上で30分間、抗体と共にインキュベートし、2%のウシ胎児血清及び1μMのアジ化ナトリウムを含むPBSによって洗浄し、次に、FITC(フルオレセインイソチオシアネート(isotiocianate))又はPE(フィコエリトリン)を追加した。フローサイトメトリーによる分析を、CELLQuestソフトウェア(Becton、Dickson and Company(米国))を使用して、FACS(蛍光活性化細胞選別装置(Becton、Dickson and Company(米国))にて実行する。
神経分化のために、本発明にかかる細胞を、1週間、20%のノックアウト血清(インビトロゲン会社(米国))、100ユニット/mLのペニシリン、100μg/mLのストレプトマイシン及び2mMのL-グルタミンを補充したDMEM培地を含む25cm2の培養瓶においてコンフルエンを維持する。従って、これらを、0.05%のトリプシン/EDTA溶液を用いて回収し、同じ培養タイプを含む35cm2のシャーレに高密度で平板播種する。神経分化は、全トランスレチノイン酸(RA)(シグマ社、米国)の添加によっても誘導される。トリプシン処理によって得られた本発明にかかる細胞の懸濁液を、35cm2のシャーレに移して、B27を補充した神経基礎培地(インビトロゲン社、米国)を含む0.1%のアガロース溶液(シグマ社、米国)で前処理する。24時間後、細胞は、球状構造を形成し、神経分化は、終濃度がそれぞれ10-7M及び0.05%のRA(レチノイン酸)及びDMSO(ジメチルスルホキシド)の追加によって誘導される。上記培地は毎日交換する。非接着条件下での4日間の培養の後、プレート上のSLS凝着を、適切な培地を含む0.1%のゼラチンで処理する。
Claims (13)
- 多能性幹細胞及び多能性前駆細胞を生産するためのプロセスであって、
A- 1又は複数の天然幹細胞ニッチ(NSCN)の取得ステップ;
B- 1又は複数のNSCNの前調製ステップ;
C- 1又は複数のNSCNの組織断片の調製ステップ;
D- 基礎培地においてNSCN断片を培養することによる幹細胞(SC)及び前駆細胞増殖の促進ステップ;
E- NSCN断片からのSCの分離ステップ;
F- 任意に、Eにおける分離されたNSCNをステップDに戻すステップを含む、プロセス。 - 前記NSCNは、1又は複数の、出産由来の胚外組織、神経、筋肉、脂肪、骨、皮膚並びに肝臓、肺、心臓、脾臓、肝臓、膵臓、精巣、卵巣、生検由来組織及びNSCNを有するがん組織といった臓器由来組織である、請求項1に記載のプロセス。
- 前記NSCNは、臍帯、胎盤又は羊膜のうちの1又は複数である、請求項1に記載のプロセス。
- ステップBの前記NSCN前調製は、清浄、洗浄、組織の事前切断、磨砕、圧縮及び解離酵素を用いたマイルドな前処理のうちの1又は複数を含む、請求項1に記載の方法。
- ステップCにおける前記断片化は、完全なNSCN組織になされる、請求項1に記載の方法。
- ステップDの前記培地は、NSCNに含まれる細胞成長に必要な栄養分、特にアミノ酸、タンパク質、血清及び抗生物質を含む、請求項1に記載のプロセス。
- ステップDの前記培地は、酸性からアルカリ性へのpH変化を防ぐために必要な頻度で変えられる、請求項1に記載のプロセス。
- ステップEの断片は、機械的手段によって取り除かれる、請求項1に記載のプロセス。
- ステップEの前記幹細胞及び前駆細胞を、組織断片から分離して、洗浄し、そして、70%から90%の半コンフルエント状態に達した後に取り除く、請求項1に記載のプロセス。
- 前記ステップEの後に分離され、前記ステップDに戻るNSCN断片は、ステップA、B及びCに従って得られた断片と混合することができる、請求項1に記載のプロセス。
- 請求項1〜10のいずれかに記載のプロセスによって取得される、幹細胞及び/又は前駆細胞。
- 請求項1〜10のいずれかに記載のプロセスによって取得される、NSCN組織断片である、請求項1に記載のNSCN組織断片。
- 請求項1〜10のいずれかに記載のプロセスによって取得される、幹細胞、前駆細胞及びNSCN組織断片。
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