JP6169972B2 - 幹細胞分離方法 - Google Patents
幹細胞分離方法 Download PDFInfo
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- JP6169972B2 JP6169972B2 JP2013542935A JP2013542935A JP6169972B2 JP 6169972 B2 JP6169972 B2 JP 6169972B2 JP 2013542935 A JP2013542935 A JP 2013542935A JP 2013542935 A JP2013542935 A JP 2013542935A JP 6169972 B2 JP6169972 B2 JP 6169972B2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Sustainable Development (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Description
ヒトまたはその他の動物から体液を採取し、ヘパリンを0.1〜500IU/mL程度添加して混和する。次に、クエン酸を0.01〜10%(w/v)程度添加し、混和する。
該細胞分離フィルターからなる細胞分離カラムに、1)で調製した体液を通液し、目的とする幹細胞を含む細胞画分を細胞分離フィルターに捕捉する。通液する際には、体液を入れた容器から送液回路を通じて自然落下で送液しても、ポンプにより通液しても良い。また体液を入れたシリンジを直接、該カラムに接続し、手でシリンジを押して注入しても良い。ポンプにより通液する場合は流速0.1〜100mL/min程度が挙げられる。
該細胞分離カラムに、流入口側から洗浄液を通液し、赤血球等の細胞を洗い流す。洗浄液は、回路を通じて自然落下で送液しても、ポンプにより通液しても良い。ポンプにより通液する場合の流速は、0.1〜100mL/min程度が挙げられる。洗浄量は、細胞分離カラムの容量により異なるが、該カラム容積の約1〜100倍程度量で洗浄することが好ましい。細胞洗浄液としては、生理的食塩液、リンゲル液、細胞培養に使用する培地、リン酸緩衝液等の一般的な緩衝液等が挙げられるが、安全面から生理的食塩液が好ましい。
該細胞分離カラムに、体液および洗浄液を流した方向とは逆方向(流出口側)から細胞回収液を入れ、目的とする幹細胞を含む画分を回収する。細胞回収液を細胞分離カラムに注入する時は、細胞回収液をシリンジ等に予め入れておき、シリンジのプランジャーを手等で勢いよく押し出すこと等により実現できる。回収液量および流速は、カラム容量により異なるが、カラム容積の1〜100倍量程度の細胞回収液を、流速0.5〜20mL/sec程度で注入することが好ましい。
(1)体液の調製
ヒト骨髄液(AllCells社製Fresh Whole Bone Marrow、ヘパリン50IU/mL含有)に、ACD−A液を骨髄液:ACD−A液=72:1の割合で添加し(クエン酸終濃度:0.028%)、十分に転倒混和を行った。
出入口を供えた内径2.2cm、長さ0.9cmの円筒状のポリカーボネイト製の筒に、細胞分離フィルターとしてレーヨンとポリオレフィンからなる不織布(目付け=95(g/m2)、繊維径=15±9μm、密度3.8×105(g/m3))を直径2.2cmの円形に打抜いて24枚を圧縮・積層し、上下をストッパーで挟み込んで細胞分離フィルターを固定した細胞分離カラムを作製した。
50mLの生理食塩液にて不織布の洗浄を行った。次に、シリンジポンプにて骨髄液3mL相当量を流速6mL/minで通液した。次に同方向から生理食塩液30mLを同流速にて流すことにより、赤血球等の洗浄除去を行った。次にウシ胎児血清10%を含む細胞培養液(GIBCO α−MEM培地)50mLを、骨髄液を流した方向と逆方向から勢い良く流すことにより、目的とする細胞画分を回収した。
回収した細胞懸濁液に含まれる白血球、赤血球、血小板数を自動血球計測装置(シスメックス K−4500)にて測定した。この結果をカラム通過前の各種細胞数で割ることにより細胞混入率を求めた。その結果、白血球混入率=62%、赤血球混入率=3%、血小板混入率=14%であった。
骨髄液通液時に、カラム入口側の圧力を圧力計で測定した。この結果を詰まりの指標とした(カラムが詰まるほど、骨髄液の通液性が損なわれてカラム入口側の圧力が上昇する)。なお本評価は、30mL骨髄液処理時(実施例4および比較例2)について行った。
CFU−Fアッセイにより間葉系幹細胞分離数を解析した。まず回収した細胞懸濁液の1/3量(骨髄液1mL処理相当分)にウシ胎児血清10%を含む細胞培養液(GIBCOα−MEM培地)を加えてポリスチレンシャーレ(直径10cm)に移し、37℃、5%CO2環境下で培養を行った。次に、培養9日後にクリスタルバイオレット−メタノール液にてコロニーを染色し、出現したコロニー数を測定したところ、回収されたコロニー数=361個であった。
ACD−A液の添加量を骨髄液:ACD−A液=100:15(クエン酸終濃度:0.28%)とした以外は、実施例1と同様の方法で、細胞混入率、コロニー数を求めた。その結果、白血球混入率=44%、赤血球混入率=3%、血小板混入率=0%、回収されたコロニー数=391個であった。
ACD−A液の添加量を骨髄液:ACD−A液=2:1(クエン酸終濃度:0.72%)とした以外は、実施例1と同様の方法で、細胞混入率、コロニー数を求めた。その結果、白血球混入率=44%、赤血球混入率=3%、血小板混入率=0%、回収されたコロニー数=401個であった。
骨髄液の処理量を30mLに、また<CFU−Fアッセイ>に用いる細胞懸濁液の量を1/30量(骨髄液1mL処理相当分)とした以外は、実施例1と同様の方法で、細胞混入率、コロニー数を求めた。その結果、白血球混入率=16%、赤血球混入率=1%、血小板混入率=5%、回収されたコロニー数=342個であった。また骨髄液通液時に、詰まりの指標としてカラム入口側の圧力を圧力計で測定したところ(カラムが詰まるほど、骨髄液の通液性が損なわれてカラム入口側の圧力が上昇する)、最大圧力は31mmHgであった。
局方クエン酸ナトリウムを生理食塩液に溶かしてブタ骨髄液(ヘパリン50IU/mL含有)にクエン酸終濃度:0.05%となるように添加して、十分に転倒混和を行った。細胞分離カラムの作製および評価については実施例1と同様の方法で、細胞混入率、コロニー数を求めた。その結果、白血球混入率=64.4%、赤血球混入率=3%、血小板混入率=0%、回収されたコロニー数=301個であった。
クエン酸終濃度を0.28%とした以外は、実施例5と同様の方法で、細胞混入率、コロニー数を求めた。その結果、白血球混入率=57.2%、赤血球混入率=3%、血小板混入率=0%、回収されたコロニー数=309個であった。
クエン酸終濃度を0.50%とした以外は、実施例5と同様の方法で、細胞混入率、コロニー数を求めた。その結果、白血球混入率=57.2%、赤血球混入率=3%、血小板混入率=0%、回収されたコロニー数=283個であった。
クエン酸終濃度を1.00%とした以外は、実施例5と同様の方法で、細胞混入率、コロニー数を求めた。その結果、白血球混入率=57.2%、赤血球混入率=3%、血小板混入率=0%、回収されたコロニー数=251個であった。
クエン酸終濃度を2.51%とした以外は、実施例5と同様の方法で、細胞混入率、コロニー数を求めた。その結果、白血球混入率=64.4%、赤血球混入率=6%、血小板混入率=0%、回収されたコロニー数=262個であった。
クエン酸終濃度を3.69%とした以外は、実施例5と同様の方法で、細胞混入率、コロニー数を求めた。その結果、白血球混入率=50%、赤血球混入率=3%、血小板混入率=0%、回収されたコロニー数=175個であった。
ACD−A液を体液に添加せずにカラム処理をした以外は、実施例1と同様の方法で、細胞混入率、コロニー数を求めた。その結果、白血球混入率=62%、赤血球混入率=3%、血小板混入率=21%、回収されたコロニー数=363個であった。
ACD−A液を体液に添加せずにカラム処理をした以外は、実施例4と同様の方法で、細胞混入率、コロニー数を求めた。その結果、白血球混入率=35%、赤血球混入率=1%、血小板混入率=29%、回収されたコロニー数=265個であった。また骨髄液通液時に、詰まりの指標としてカラム入口側の圧力を圧力計で測定したところ、最大圧力は92mmHgであった。
Claims (9)
- 下記(1)〜(2)の工程:
(1)ヘパリンを含有する体液にクエン酸を添加する工程、および
(2)細胞分離フィルターを用いて、該体液から付着性の幹細胞を分離する工程、
をこの順で行う幹細胞の分離方法であって、
クエン酸の添加濃度が0.05%(w/v)以上であることを特徴とする、幹細胞の分離方法。 - 幹細胞が間葉系幹細胞であることを特徴とする、請求項1に記載の方法。
- 体液が骨髄液であることを特徴とする、請求項1または2に記載の方法。
- クエン酸の添加濃度が3.69%(w/v)以下であることを特徴とする、請求項1〜3のいずれかに記載の方法。
- 分離された幹細胞を含む細胞懸濁液中の血小板の混入率が20%以下であることを特徴とする、請求項1〜4のいずれかに記載の方法。
- 細胞分離フィルターがポリオレフィンまたは/およびレーヨンからなることを特徴とする、請求項1〜5のいずれかに記載の方法。
- 細胞分離フィルターが不織布からなることを特徴とする、請求項1〜6のいずれかに記載の方法。
- 不織布の密度が2.1×105〜4.9×105g/m3であり、かつ、繊維径が3〜40μmであることを特徴とする、請求項7に記載の方法。
- 細胞分離フィルターが体液の流入口と流出口を有する容器に充填されていることを特徴とする、請求項6〜8のいずれかに記載の方法。
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