JP5312804B2 - 幹細胞及び胎児由来の心筋細胞及び予定心筋細胞の精製方法 - Google Patents
幹細胞及び胎児由来の心筋細胞及び予定心筋細胞の精製方法 Download PDFInfo
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Description
Muller M, et al., FASEB J. 2000; 14: 2540-2548 Klug MG, et al., J. Clin. Invest. 1996; 98: 216-224 Schroder AR, et al., Cell. 2002; 110: 521-529 Khairallah M, et al., Am J Physiol Heart Circ Physiol 2004; 286, H1461-1470 Chatham JC, et al., J Biol Chem 1995; 270: 7999-8008
(1)低・無血清条件において非心筋細胞に志向性を有する細胞分化増殖抑制並びに細胞死誘導が起こる現象;
(2)弱酸性培養液における非心筋細胞に志向性を有する細胞死誘導が起こる現象;
(3)低カルシウム培養液における非心筋細胞に志向性を有する細胞分化増殖抑制並びに細胞死誘導が起こる現象;
(4)低栄養培養液で培養した場合に非心筋細胞が選択的に死亡する現象;
(5)低カルシウム環境で心筋細胞を培養した場合に心筋細胞の自律拍動が減弱し、エネルギー消費が抑制される現象;
(6)低・無血清化が心筋細胞の自己集合塊形成を促進する現象;及び
(7)心筋胞細胞塊をバラバラに分散した場合心筋細胞の生存率が著しく低下する現象;
を見出した。
本実施例においては、心筋細胞を含む細胞塊(胚様体)を、血清を除去した培養液中で培養することにより、心筋細胞を含む細胞塊(胚様体)に対する血清枯渇の作用、より具体的には細胞塊(胚様体)から心筋細胞を選択する際における血清枯渇の作用、について検討することを目的とした。
本実施例においては、予定心筋細胞を含む細胞塊(胚様体)を、血清を除去した培養液中で培養することにより、予定心筋細胞を含む細胞塊(胚様体)に対する血清枯渇の作用、より具体的には細胞塊(胚様体)から予定心筋細胞を選択する際における血清枯渇の作用、について検討することを目的とした。
本実施例においては、心筋細胞を含む細胞塊(胚様体)を、カルシウム濃度を低下させた培養液中で培養することにより、心筋細胞を含む細胞塊(胚様体)に対するカルシウム濃度の低下の作用、より具体的には細胞塊(胚様体)から心筋細胞を選択する際におけるカルシウム濃度の低下の作用、について検討することを目的とした。
本実施例においては、予定心筋細胞を含む細胞塊(胚様体)を、血清を除去し、かつ弱酸性である培養液中で培養することにより、予定心筋細胞を含む細胞塊(胚様体)に対する血清枯渇および弱酸性pHの複合的な作用、より具体的には細胞塊(胚様体)から予定心筋細胞を選択する際における血清枯渇および弱酸性pHの複合的な作用、について検討することを目的とした。
本実施例においては、心筋細胞を含む細胞塊(胚様体)を、血清を除去し、かつ弱酸性である培養液中で培養することにより、心筋細胞を含む細胞塊(胚様体)に対する血清枯渇および弱酸性pHの複合的な作用、より具体的には細胞塊(胚様体)から心筋細胞を選択する際における血清枯渇および弱酸性pHの複合的な作用、について検討することを目的とした。
本実施例においては、心筋細胞を含む細胞塊(胚様体)を、血清を除去し、かつ糖類を除去した培養液中で培養することにより、心筋細胞を含む細胞塊(胚様体)に対する血清枯渇および糖類枯渇の複合的な作用、より具体的には細胞塊(胚様体)から心筋細胞を選択する際における血清枯渇および糖類枯渇の複合的な作用、について検討することを目的とした。
実施例6にて作製した細胞塊には、死細胞や細胞外マトリックスが付着している。これを除去し心筋細胞塊のみを精製する必要がある。しかしながら、事前の検討において、トリプシン等のタンパク質を非選択的に消化する酵素を用いた場合、心筋細胞の生存率は著しく低下することが明らかになった。
本実施例においては、心筋細胞を含む細胞塊(胚様体)を、血清を除去し、弱酸性であり、カルシウムを除去し、かつ糖類を除去した培養液中で培養することにより、心筋細胞を含む細胞塊(胚様体)に対する血清枯渇、弱酸性pH、低カルシウム、および糖類枯渇の複合的な作用、より具体的には細胞塊(胚様体)から心筋細胞を選択する際における血清枯渇、弱酸性pH、低カルシウム、および糖類枯渇の複合的な作用、について検討することを目的とした。
本実施例においては、心筋細胞を含む細胞塊(胚様体)を、血清を除去し、弱酸性であり、カルシウムを除去し、かつ糖類を除去した培養液中にアスパラギン酸・グルタミン酸を添加して培養することにより、心筋細胞を含む細胞塊(胚様体)に対するアスパラギン酸・グルタミン酸の代償性の作用、より具体的には細胞塊(胚様体)から心筋細胞を選択する際におけるアスパラギン酸・グルタミン酸の代償性の作用、について検討することを目的とした。
本実施例においては、マウス胎児から、心筋細胞を精製することを目的として行った。
本実施例においては、心筋細胞を含む細胞塊(胚様体)を、血清を除去し、弱酸性であり、カルシウムを除去し、かつ糖類を除去した培養液中にピルビン酸を添加して培養することにより、心筋細胞を含む細胞塊(胚様体)に対するピルビン酸の代償性の作用、より具体的には細胞塊(胚様体)から心筋細胞を選択する際におけるピルビン酸の代償性の作用、について検討することを目的とした。
本実施例においては、霊長類マーモセット胚性幹細胞由来心筋細胞を含む細胞塊(胚様体)を、血清を除去し、かつ糖類を除去した培養液中で培養することにより、心筋細胞を含む細胞塊(胚様体)に対する血清枯渇および糖類枯渇の複合的な作用、より具体的には霊長類マーモセット胚性幹細胞由来の細胞塊(胚様体)から心筋細胞を選択する際における血清枯渇および糖類枯渇の複合的な作用、について検討することを目的とした。
本実施例においては、霊長類マーモセット胚性幹細胞由来心筋細胞の利用方法である心臓に対する移植と生着に関する検討を目的とした。
本実施例においては、ヒト胚性幹細胞由来心筋細胞を含む細胞塊(胚様体)を、血清を除去し、かつ糖を除去した培養液中で培養することにより、心筋細胞を含む細胞塊(胚様体)に対する血清枯渇および糖枯渇の複合的な作用、より具体的にはヒト胚性幹細胞由来の細胞塊(胚様体)から心筋細胞を選択する際における血清枯渇および糖枯渇の複合的な作用、について検討することを目的とした。
Claims (13)
- 胚性幹細胞由来、成体幹細胞由来又は胎児由来の心筋細胞と非心筋細胞とを含む細胞混合物から、心筋細胞を選択する方法であって、前記細胞混合物を、
(i)糖類を含まない条件または分化誘導時に用いた培養液中の糖類の条件と比較して糖類を1%未満まで低下させた条件である低糖条件;および
(ii)培養液中のカルシウム濃度が0.3〜1.3 mMの条件である低カルシウム条件、培養液に含有されるすべての栄養成分が、当該培養液中の栄養成分と比較して10%以下まで低下している条件である低栄養条件、乳酸添加条件、アスパラギン酸・グルタミン酸添加条件、およびピルビン酸添加条件、からなる群から選択される1または複数の条件;
を有する培養液中で培養することを特徴とする、心筋細胞を選択する方法。 - 更にコラゲナーゼを用いて心筋細胞に付着する死細胞を除去することからなる、請求項1に記載の心筋細胞を選択する方法。
- 心筋細胞の比重と比較して、死細胞の比重が高比重であることを利用して、死細胞を除去する、請求項2に記載の心筋細胞を選択する方法。
- 培養液の(ii)の条件が乳酸添加条件である、請求項1〜3のいずれか1項に記載の心筋細胞を選択する方法。
- 培養液の(ii)の条件がアスパラギン酸・グルタミン酸添加条件である、請求項1〜3のいずれか1項に記載の心筋細胞を選択する方法。
- 培養液の(ii)の条件がピルビン酸添加条件である、請求項1〜3のいずれか1項に記載の心筋細胞を選択する方法。
- 心筋細胞と非心筋細胞とを含む細胞混合物を、胚性幹細胞に対して分化誘導して、Nkx2.5陰性かつBrachyury陽性の細胞を含む胚様体を形成し、この胚様体を血清を含まない条件、またはNkx2.5陰性かつBrachyury陽性の細胞を得るまでの段階において培養液に添加した血清または血清成分の濃度を100%として算出した場合に血清または血清成分の濃度が0%〜10%の条件である低血清条件およびpH6.5〜7の範囲のpH条件の培養液中で培養することにより調製する、請求項1に記載の心筋細胞を選択する方法。
- 胚性幹細胞由来の心筋細胞を選択する方法であって、胚性幹細胞に分化誘導してNkx2.5陰性かつBrachyury陽性の細胞を含む胚様体を形成した後、この胚様体を血清を含まない条件、またはNkx2.5陰性かつBrachyury陽性の細胞を得るまでの段階において培養液に添加した血清または血清成分の濃度を100%として算出した場合に血清または血清成分の濃度が0%〜10%の条件である低血清条件およびpH6.5〜7の範囲のpH条件の培養液中で培養することにより予定心筋細胞を含む細胞混合物を調製し、そして同一の培養液中で当該細胞混合物の培養を継続することにより心筋細胞を得ることを特徴とする、前記心筋細胞を選択する方法。
- 培養液が、RPMI培養液、DMEM培養液、MEM培養液、F12培養液およびα-MEM培養液からなる群から選択される、請求項1〜6のいずれか1項に記載の心筋細胞を選択する方法。
- 乳酸添加条件が、乳酸を0.1〜5 mM添加する条件である、請求項1〜6のいずれか1項に記載の心筋細胞を選択する方法。
- アスパラギン酸・グルタミン酸添加条件が、アスパラギン酸を20〜100 mg/L、グルタミン酸を20〜100 mg/L添加する条件である、請求項1〜6のいずれか1項に記載の心筋細胞を選択する方法。
- ピルビン酸添加条件が、ピルビン酸を0.5〜5 mM添加する条件である、請求項1〜6のいずれか1項に記載の心筋細胞を選択する方法。
- pH条件がpH6.5の条件である、請求項7または8に記載の心筋細胞を選択する方法。
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