JP7441447B2 - ヘルニア嚢から間葉系幹細胞を単離精製する方法、及び組織修復するためのヘルニア嚢由来の間葉系幹細胞の使用 - Google Patents
ヘルニア嚢から間葉系幹細胞を単離精製する方法、及び組織修復するためのヘルニア嚢由来の間葉系幹細胞の使用 Download PDFInfo
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Description
本明細書に記載の数値は、概算値である。全ての実験データは、その数値の±20%の範囲、好ましいは±10%の範囲、より好ましくは±5%の範囲を示す。
成人鼠蹊部ヘルニア(inguinalhernia)の手術(図1のA、Bは、いずれも手術中のヘルニア嚢を示す。)は、鼠径部を切開することによる開放式手術を含む。ヘルニア手術で無作為に選択した4人の患者から新鮮なヘルニア嚢(herniasac)を採取し、すぐに実験室に運ぶ。表1には、この4人の患者の情報が掲載されている。48時間以内に間葉系幹細胞(mesenchymal stem cell、MSC)を採取する。
国際細胞治療学会(International Society for Cellular Therapy)により、下記(1)~(3)に記載の多能性間葉系幹細胞の最低基準に基づいて、間葉系幹細胞の存在を確認する。
(2)特異的な表面抗原(Ag)の発現。
(3)多能性分化の可能性。
本実施例は、従来文献であるM. Dominici, K. Le Blanc, I. Mueller, et al., Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement, Cytotherapy, 8 (4) (2006), pp. 315-317)、及びヒト間葉系幹細胞の抗体の利用可能性に基づいて陽性マーカーを選択する。CD105、CD73、及びCD90マーカーは、ヒト間葉系幹細胞の発現が95%以上であることを評価するために用いられる。CD45、CD34、CD11b、CD19、及びヒト白血球抗原-DR(human leukocyte antigen-DR、HLA-DR)マーカーは、ヒト間葉系幹細胞の発現を欠いている(2%以下)ことを評価するために用いられる。評価結果として、使用された全ての陽性CDマーカー(CD105、CD73及びCD90)の発現は、いずれも95%以上である。
間葉系幹細胞が90%の密集度に成長すると、培地を3種類の分化培地に移して三血球系(trilineage)の能力を測定する。分化培地は、それぞれStemMACS Osteodiff培地、StemMACS Chondrodiff培地、又はStemMACS Adipodiff培地(Miltenyi Biotec)である。骨形成(osteogenesis)を検証するために、培地をStemMACS Osteodiff培地に移し、Von Kossaで染色する。
B方法は、よく論文で見られる方法であり、異なる組織を処理した後の細胞数を定量的に比較することができる方法である。ヘルニア嚢組織には血管が豊富に存在するため、取得した間質血管分画(stromal vascular fraction、SVF)細胞は、同じ処理によって皮下脂肪組織から取得したSVF細胞と比べて、有意に多くのコロニー形成単位(colony forming unit、CFU)を有する(図6参照)。前記CFUは、間葉系幹細胞のコロニーに相当する。よって、本実施例の結果から分かるように、同じ方法で、ヘルニア嚢組織から得られた間葉系幹細胞は、脂肪組織から得られた間葉系幹細胞と比べて、細胞数が多く、かつ健康である(細胞分裂回数が多い)。
図7は、皮下脂肪組織由来の間葉系幹細胞と比べて、ヘルニア嚢由来の間葉系幹細胞がより多くの細胞分裂回数を有し、累積細胞数がより多いことを示している。そのため、本発明の方法で単離精製したヘルニア嚢由来の間葉系幹細胞は、高い細胞分裂能力を有する。
Claims (12)
- (a)ヘルニア嚢のサンプルを組織切片に切断する工程と、
(b)前記組織切片を洗浄して第1所定時間で静置し、上清及び洗浄された組織切片を形成した後、前記上清を除去する工程と、
(c)第1培地中で前記洗浄された組織切片に対して順に均質処理、再懸濁処理を行うことにより、間葉系幹細胞を含む混合物を形成し、前記間葉系幹細胞を含む混合物を濾過して濾過物を得て、前記濾過物を培養フラスコに播種し、第2所定時間で培養する工程と、
(d)培養フラスコに付着した前記間葉系幹細胞を単離し、第2培地を添加して第3所定時間で連続培養することにより、前記間葉系幹細胞を精製する工程と、を含み、
工程(b)において、酵素処理を行う必要がないことを特徴とする、
ヘルニア嚢から間葉系幹細胞を単離精製する方法。 - 前記組織切片の体積は、1~2mm3であることを特徴とする、
請求項1に記載の方法。 - 前記第1所定時間は、4~6分であることを特徴とする、
請求項1に記載の方法。 - 工程(b)を2回以下繰り返すことを特徴とする、
請求項1に記載の方法。 - 前記第1培地及び前記第2培地は、いずれもα-最小必須培地(alpha-minimal essential medium)であることを特徴とする、
請求項1に記載の方法。 - 前記第1培地又は前記第2培地に10%のFBS(fetal bovine serum、FBS)、及び1%のペニシリン(penicillin)及びストレプトマイシン(streptomycin)を含む溶液をさらに添加することを特徴とする、
請求項5に記載の方法。 - 前記第2所定時間は、24~48時間であることを特徴とする、
請求項1に記載の方法。 - 工程(d)において、トリプシン(trypsin)によって培養フラスコに付着した前記間葉系幹細胞を単離することを特徴とする、
請求項1に記載の方法。 - 前記第3所定時間は、7~10日であることを特徴とする、
請求項1に記載の方法。 - 前記ヘルニア嚢は、鼠蹊部由来のものであることを特徴とする、
請求項1に記載の方法。 - (i)請求項1~10のいずれか1項に記載の方法によってヘルニア嚢由来の間葉系幹細胞を単離精製する工程と、
(ii)前記工程(i)により単離精製されたヘルニア嚢由来の間葉系幹細胞を組織修復用の医薬品を製造するために使用する工程と、を含むことを特徴とする、
組織修復用の医薬品を製造するためのヘルニア嚢由来の間葉系幹細胞の使用。 - 前記組織修復は、筋肉組織の修復であることを特徴とする、
請求項11に記載の使用。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5486359A (en) | 1990-11-16 | 1996-01-23 | Osiris Therapeutics, Inc. | Human mesenchymal stem cells |
JP2007509601A (ja) | 2003-11-04 | 2007-04-19 | 株式会社バイオマスター | 脂肪組織から幹細胞を調製するための方法およびシステム |
WO2009034708A1 (ja) | 2007-09-11 | 2009-03-19 | Sapporo Medical University | 細胞増殖方法ならびに組織の修復および再生のための医薬 |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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US5486359A (en) | 1990-11-16 | 1996-01-23 | Osiris Therapeutics, Inc. | Human mesenchymal stem cells |
JP2007509601A (ja) | 2003-11-04 | 2007-04-19 | 株式会社バイオマスター | 脂肪組織から幹細胞を調製するための方法およびシステム |
WO2009034708A1 (ja) | 2007-09-11 | 2009-03-19 | Sapporo Medical University | 細胞増殖方法ならびに組織の修復および再生のための医薬 |
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The hernia sac - A suitable source for obtaining mesenchymal stem cells,2021年,Vol.6,p.40-44 |
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