JP2015532596A - 間葉系幹細胞を培養するための方法 - Google Patents
間葉系幹細胞を培養するための方法 Download PDFInfo
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Abstract
Description
カルシウム濃度による増殖能を調べるために、臍帯血由来間葉系幹細胞を種々の濃度のカルシウムの存在下で培養した。
酸素濃度による増殖能を調べるために、臍帯血由来間葉系幹細胞を種々の濃度の酸素の存在下で培養した。
カルシウム(マグネシウムを含む。)と酸素濃度状態との組み合わせによる、臍帯血由来間葉系幹細胞の増殖能の試験を行った。細胞を典型的状態(対照)、外添したカルシウム(マグネシウムを含む。)の存在下、低酸素状態、及び外添したカルシウム(マグネシウムを含む)/低酸素状態(以下「CMH」と呼ぶ。)で培養した。これに関して、培地は、カルシウム及びマグネシウムをそれぞれ3.6及び1.8Mの総濃度で含有した(1.8mMカルシウム及び1mMマグネシウムを追加的に添加)。低酸素状態は酸素レベル3%に設定した。細胞を実施例1に類似の方法で培養した。典型的状態での5継代(P5)後、間葉系幹細胞を、継代間7日の一定の間隔でCMH状態下7回の継代(P12)により培養した。
臍帯血由来間葉系幹細胞の生存率及び回収率に対する本発明のCMH状態の影響の試験を行った。このために、臍帯血由来間葉系幹細胞(MSC #1)を典型的状態(対照)、低酸素状態(3%)、増加カルシウム状態(1.8mM;培地中3.6mMの総カルシウムレベル)、及びCMH状態(3%O2+1.8mM添加カルシウム+1mM添加マグネシウム)で培養し、培養容器から剥離し、基礎培地(α−MEM)で3回洗浄し、基礎培地(α−MEM)に懸濁した。室温で維持しながら、細胞懸濁液を生存率及び回収率について経時的に調べた。細胞生存率は、回収して基礎培地に懸濁した細胞をトリパンブルーで染色し、所定容量(10〜20μL)の懸濁液中の青く染色された生細胞を含む全細胞を、血球計算盤を用いてカウントした後、死細胞に対する生細胞の割合(%)として表した。回収率は、培養前に対する培養後の生細胞数の割合(%)として表した。
なぜCMH状態が臍帯血由来間葉系幹細胞の増殖を改善するのかを調べるために、幹細胞の増殖に関連する該細胞の幹細胞性及び老化をアッセイした。
臍帯血由来間葉系幹細胞の特性に対するCMH状態の影響の試験を行った。この目的のために、間葉系幹細胞を、軟骨形成誘導及び骨形成誘導による分化能及びマーカー発現についてアッセイした。
軟骨形成誘導における使用のために、細胞を2〜2.5×105細胞の集団で15mLコニカルチューブに入れ、遠心分離して細胞ペレットを得た。細胞ペレットをD−PBSで洗浄し、200〜250μlの分化培地[高グルコースDMEM(Gibco, cat#.11995)、10ng/ml TGFβ−3(Sigma, cat#.T5425, 2μg)、500ng/ml BMP−6(R&D, cat#.507−BP, 20μg)、50μg/mlアスコルビン酸(Sigma, cat#.A8960)、50mg/ml(1:100)ITS(商標)+Premix(BD, cat#.354352)、40μg/ml L−プロリン(Sigma, cat#.P5607)、100μg/mlピルビン酸ナトリウム(Sigma, cat#.P8574)、100nMデキサメタゾン(Sigma, cat#.D2915)]に懸濁し、細胞懸濁液をチューブに等分した。これらのチューブを1500rpmで5分間遠心分離し、その後、細胞を37℃のCO2インキュベーターにおいて、チューブを僅かに開いた状態で4週間培養して軟骨への分化を誘導した。分化培地は週に2回、新鮮なものと半分のみ取り替えた。
軟骨形成誘導後、細胞を遠心分離し、PBSで洗浄し、4%パラホルムアルデヒド中、室温で0.5〜1時間固定した。その後、細胞を蒸留水で2回又は3回洗浄し、凍結切片法を用いて切片(4〜5μm厚)に調製した。切片を95%エタノールに3〜5分間浸漬させ、水で2回洗浄した。0.1%サフラニンOで7分間染色した後、細胞を70%エタノールで2回、70%エタノールで1回、95%エタノールで2回、95%エタノールで1回、及び100%エタノールで2回洗浄し、キシレン基質溶液に3分間浸漬し、乾燥させた。その後、染色細胞を脂溶性封入溶液で覆い、観察した。軟骨形成誘導を、色(紫)、分化したペレットのサイズ、及び形成された小腔構造を比較して評価した。
骨形成誘導における使用のために、細胞を密度500〜1000細胞/ウェルで6ウェルプレートに播種し、2〜4日後、培地を骨形成誘導培地(1Lのα−MEM中、β−グリセロールリン酸 2.1604g、L−アスコルビン酸−2−ホスフェート 0.012805g、デキサメタゾン/UVAB 0.6mg、ゲンタマイシン(10mg/ml) 5ml、及びFBS 100ml)と取り替えた。細胞を、3日ごとに新鮮なものと取り替えた分化培地を用いて2〜3週間培養した。軟骨形成誘導をALP染色法により評価した。
分化細胞をPBSで2回洗浄し、固定液(40%アセトン)で30〜45秒間インキュベートした。分化細胞を蒸留水で2回又は3回再び洗浄し、暗所でアルカリ染色溶液(ファストバイオレットB塩)と30分間インキュベートした。次いで、細胞を蒸留水で2回洗浄し、マイヤーヘマトキシリン溶液により10〜20秒間処理した。染色溶液の除去後、細胞を水道水で洗浄し、乾燥させ、脂溶性封入溶液で覆い、観察した。骨芽細胞は細胞内アルカリホスファターゼの活性化により暗褐色に染色されるため、軟骨形成誘導を染色の程度により評価した。
培養状態に応じた臍帯血由来間葉系幹細胞の免疫学的特性を、次のように混合リンパ球反応(MLR)を用いて評価した。
臍帯血由来間葉系幹細胞のサイトカイン放出能力に対する培養状態の影響を、臍帯血由来間葉系幹細胞が軟骨細胞に分化する間に放出されるTsp−2を測定することによってアッセイした。
Claims (13)
- 間葉系幹細胞を培養するための方法であって、2〜5%酸素の低酸素状態下、2.1〜3.8mMの濃度のカルシウム及び1.0〜3.0mMの濃度のマグネシウムを含有する培地で間葉系幹細胞を培養するステップを含む方法。
- 間葉系幹細胞は臍帯血、骨髄、脂質、筋肉、皮膚、羊水、臍帯、又は歯に由来する、請求項1に記載の方法。
- 培地は、ダルベッコ改変イーグル培地(DMEM)、最小必須培地(MEM)、α−MEM、マッコイ5A培地、イーグル基礎培地、CMRL(Connaught Medical Research Laboratory)培地、グラスゴーMEM、Ham’s F−12培地、IMDM(イスコフ改変ダルベッコ培地)、Leibovitz’s L−15 培地、RPMI(Roswell Park Memorial Institute)1640培地、199培地、及びハンクス199培地からなる群から選択される、請求項1に記載の方法。
- 培地は5〜30%のウシ胎児血清を含む、請求項3に記載の方法。
- 培地はウシ胎児血清を含まず、血清代替物を含む、請求項3に記載の方法。
- 培地は、5〜30%のウシ胎児血清(FBS)、0.3〜2.0mMのカルシウム、及び0.2〜2.2mMのマグネシウムが添加されたα−MEMをベースとするものである、請求項1に記載の方法。
- 間葉系幹細胞は請求項1と同じ状態で継代培養される、請求項1に記載の方法。
- 請求項1に記載の方法を用いて調製された間葉系幹細胞であって、増殖能、生存率、回収率、及び免疫学的特性において改善されている間葉系幹細胞。
- 請求項8に記載の間葉系幹細胞を含む細胞治療剤。
- 脂肪細胞、骨細胞、軟骨細胞、筋細胞、神経細胞、心筋細胞、肝細胞、膵島ベータ細胞、血管細胞、又は肺細胞の再生又は保護に適用される、請求項9に記載の細胞治療剤。
- 肺疾患の治療、肺疾患誘発性炎症の抑制若しくは治療、肺組織の再生、及び肺線維症の抑制からなる群から選択されるものに有用である、請求項9に記載の細胞治療剤。
- 軟骨の再生に適用される、請求項9に記載の細胞治療剤。
- 自己免疫疾患又は移植片対宿主病の治療に適用される、請求項9に記載の細胞治療剤。
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JP7336557B2 (ja) | 2016-03-31 | 2023-08-31 | サンバイオ,インコーポレイティド | 幹細胞の培養及び治療のための培地、方法、細胞、及び分泌因子 |
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JP7350314B2 (ja) | 2017-06-05 | 2023-09-26 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | 網膜疾患を処置するための組成物ならびにそれを作製および使用するための方法 |
JP2022013636A (ja) * | 2020-06-30 | 2022-01-18 | ミラセルバイオ カンパニー リミテッド | 類似間葉系幹細胞の製造方法及びこれにより製造された類似間葉系幹細胞 |
JP7069492B2 (ja) | 2020-06-30 | 2022-05-18 | ミラセルバイオ カンパニー リミテッド | 類似間葉系幹細胞の製造方法及びこれにより製造された類似間葉系幹細胞 |
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ES2755802T3 (es) | 2020-04-23 |
US20190218517A1 (en) | 2019-07-18 |
EP2893001B1 (en) | 2019-10-30 |
CA2884000A1 (en) | 2014-03-06 |
CN104781394B (zh) | 2017-12-12 |
CN104781394A (zh) | 2015-07-15 |
EP2893001A1 (en) | 2015-07-15 |
EP2893001A4 (en) | 2016-03-09 |
PL2893001T3 (pl) | 2020-03-31 |
US20150284686A1 (en) | 2015-10-08 |
WO2014035215A1 (en) | 2014-03-06 |
US20170191036A1 (en) | 2017-07-06 |
IN2015DN01737A (ja) | 2015-05-29 |
DK2893001T3 (da) | 2019-12-02 |
US9580687B2 (en) | 2017-02-28 |
KR101532556B1 (ko) | 2015-06-30 |
US10294456B2 (en) | 2019-05-21 |
AU2013309642A1 (en) | 2015-03-26 |
JP6478243B2 (ja) | 2019-03-06 |
PT2893001T (pt) | 2019-12-09 |
KR20140030703A (ko) | 2014-03-12 |
AU2013309642B2 (en) | 2018-10-25 |
US11332715B2 (en) | 2022-05-17 |
CA2884000C (en) | 2021-08-24 |
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