JP2016067312A - 軟骨分化培養液、及び軟骨組織 - Google Patents
軟骨分化培養液、及び軟骨組織 Download PDFInfo
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Abstract
Description
ビタミン液と、
EAA(Essential Amino Acids)と、
エリスロポエチンと、
コンドロイチン硫酸と、
ヒアルロン酸と、
活性型ビタミンD3と、を含む軟骨分化培養液を提供する。
(移植細胞の準備)
移植細胞としては、以下の手順によりヒト腸骨骨髄液から採取した間葉系幹細胞を用いた。
(軟骨への分化誘導)
増殖させた細胞を2×105細胞個/1mlの密度で、各実験例で調製した軟骨分化培養液を用いた軟骨分化培地に懸濁させ、15ml遠枕管に移し、500Gで遠心し、ペレット(細胞塊)を得た。
(評価方法)
上述のように各実験例の軟骨分化培養液を用いて軟骨へと分化誘導した後、以下の2つの手法により評価を行った。
<病理組織学的評価>
各実験例の軟骨分化培養液により培養した組織を10%ホルマリン中性緩衝溶液で2日間固定した後にパラフィン包埋し、ミクロトームにて厚さ5μmに切り出し、トルイジンブルー染色を行った。そして、光学顕微鏡下にて観察を行い染色の有無を確認した。
<生物学的評価>
培養した組織をパパイン水溶液で消化したサンプルをGAG定量キットおよびDNA定量キットで定量し、単位DNAあたりのグリコサミノグリカン量を算定し、別途細胞数とDNA量の関係を計測した結果より単位細胞あたりのグリコサミノグリカン量を定量した。II型コラーゲン、I型コラーゲンはELISAにより定量し、同様に単位細胞あたりの数値として算出した。
○ : Ctrl <<< Ex (実験結果(Ex)がコントロール(Ctrl)の1.2倍以上)
△ : Ctrl < Ex (実験結果(Ex)がコントロール(Ctrl)の1.05倍以上1.2倍未満)
× : Ctrl = Ex (実験結果(Ex)がコントロール(Ctrl)の1倍以上1.05倍未満)
×× : Ctrl > Ex (実験結果(Ex)がコントロール(Ctrl)未満)
いずれの実験例においても(1)単位細胞あたりのグリコサミノグリカン量についての評価と、(2)II型コラーゲン比率(II/(I+II)×100(%))についての評価とは同じ評価となった。このため、表7、表8の生物学的評価の欄で1つの結果として示している。
以下の表6に示した共通成分と、各実験例について表7に示した各成分とを混合することにより軟骨分化培養液を調製した。
上述の表6に示した共通成分と、各実験例について表8に示した各成分とを混合した点以外は、例1と同様にして軟骨分化培養液を調製した。そして、得られた軟骨分化培養液を用いて上述の手順により軟骨への分化誘導を行った後、培養した軟骨組織について上述の評価を行った。結果を表8に示す。
Claims (3)
- 脂肪酸濃縮液と、
ビタミン液と、
EAA(Essential Amino Acids)と、
エリスロポエチンと、
コンドロイチン硫酸と、
ヒアルロン酸と、
活性型ビタミンD3と、を含む軟骨分化培養液。 - 前記脂肪酸濃縮液の含有量が体積比で0.01%以上10%以下、
前記ビタミン液の含有量が体積比で0.01%以上10%以下、
前記EAAの含有量が体積比で0.01%以上10%以下、
前記エリスロポエチンの含有量が0.1IU/mL以上1000IU/mL以下、
前記コンドロイチン硫酸の含有量が0.1μg/mL以上1000μg/mL以下、
前記ヒアルロン酸の含有量が0.01μg/mL以上100μg/mL以下、
前記活性型ビタミンD3の含有量が0.01ng/mL以上100ng/mL以下である請求項1に記載の軟骨分化培養液。 - 請求項1または2に記載された軟骨分化培養液を用いた培地中で、分化した軟骨細胞からなる軟骨組織。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06312931A (ja) * | 1993-05-06 | 1994-11-08 | Teijin Ltd | 軟骨代謝異常疾患の治療剤 |
JP2007517001A (ja) * | 2003-12-30 | 2007-06-28 | ビオネトス・ホールディング・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング | 組織再生法 |
JP2008136396A (ja) * | 2006-11-30 | 2008-06-19 | Yokohama City Univ | 軟骨細胞の培養方法 |
JP2009540826A (ja) * | 2006-06-20 | 2009-11-26 | ジェンザイム・コーポレーション | 軟骨細胞増幅のための無血清培地およびその使用 |
WO2014078579A1 (en) * | 2012-11-15 | 2014-05-22 | International Stem Cell Corporation | Differentiation of human fibroblast cells |
-
2014
- 2014-09-30 JP JP2014201804A patent/JP2016067312A/ja active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06312931A (ja) * | 1993-05-06 | 1994-11-08 | Teijin Ltd | 軟骨代謝異常疾患の治療剤 |
JP2007517001A (ja) * | 2003-12-30 | 2007-06-28 | ビオネトス・ホールディング・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング | 組織再生法 |
JP2009540826A (ja) * | 2006-06-20 | 2009-11-26 | ジェンザイム・コーポレーション | 軟骨細胞増幅のための無血清培地およびその使用 |
JP2008136396A (ja) * | 2006-11-30 | 2008-06-19 | Yokohama City Univ | 軟骨細胞の培養方法 |
WO2014078579A1 (en) * | 2012-11-15 | 2014-05-22 | International Stem Cell Corporation | Differentiation of human fibroblast cells |
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