JP7109719B2 - 特定細胞捕捉方法 - Google Patents
特定細胞捕捉方法 Download PDFInfo
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Description
特定細胞捕捉方法は、採取した血液又は体液を希釈した後、前記凝集、前記遠心分離を行うものであることが好ましい。
希釈は、緩衝溶液又は液体培地を用いて行われることが好ましい。
本発明はまた、前記特定細胞捕捉方法を用いて捕捉した血液又は体液中の特定細胞を調べる特定細胞検査方法に関する。
血球細胞同士を凝集させる方法は、このような凝集が可能な方法であれば、特に限定されないが、なかでも、抗原抗体反応を利用する方法を好適に使用できる。具体的には、血球凝集反応等の凝集反応法を好適に利用できる。
(1)、(2)の保持、乾燥時間は、基材の大きさ、導入する液種、等により適宜設定すれば良い。保持時間は、5分~10時間が好ましく、10分~5時間がより好ましく、15分~2時間が更に好ましい。乾燥は、室温(約23℃)から80℃で行うことが好ましく、室温から50℃で行うことがより好ましい。また、減圧して乾燥しても良い。更に、保持して一定時間後、適宜、余分な親水性ポリマー溶液・分散液を排出し、乾燥してもよい。
AIBN(アゾビスイソブチロニトリル)を用いて、2-メトキシエチルアクリレートを80℃で6時間熱重合し、ポリ2-メトキシエチルアクリレートを作製した(分子量Mn:約15000、Mw:約50000)。そして、得られたポリ2-メトキシエチルアクリレートの1.0%メタノール溶液を作製した。
ポリスチレン製の24ウェルプレートのウェル部に、作製したポリ2-メトキシエチルアクリレート溶液(1質量%)を注入し、30分室温放置した後、液をピペットで一部吸出し、乾燥することで、医療用検査装器具を製造した。
ポリ2-メトキシエチルアクリレート溶液の濃度を2.5質量%に変更した以外は、器具例1と同様にして医療用検査器具を製造した。
ポリ2-メトキシエチルアクリレート溶液の濃度を5.0質量%に変更した以外は、器具例1と同様にして医療用検査器具を製造した。
ホウけい酸ガラス製プレートであるスライドチャンバーを用い、ポリ2-メトキシエチルアクリレート溶液の濃度を0.3質量%に変えた以外は、器具例1と同様にして医療用検査器具を製造した
ポリ2-メトキシエチルアクリレート溶液の濃度を0.5質量%に変更した以外は、器具例4と同様にして医療用検査器具を製造した。
ポリ2-メトキシエチルアクリレート溶液を注入せず、ポリ2-メトキシエチルアクリレート層を形成しなかった以外は、器具例1と同様にして医療用検査器具を製造した。
医療用検査器具の親水性ポリマー層の膜厚は、親水性ポリマー層の断面を、TEMを使用し、加速電圧15kV、1000倍で測定(撮影)した。
医療用検査器具の親水性ポリマー層の表面に蒸留水2μlを滴下し、30秒後の接触角をθ/2法(室温)で測定した。
染色をしたヒト結腸癌由来上皮細胞(HT-29)を全血に100個/血液1mLになる様に懸濁させた(スパイク血)。これに等量の液体培地を入れて希釈した(希釈スパイク血)。このあと、作製された希釈スパイク血をそのまま用いたものと、希釈スパイク血を下記の様に血球細胞同士を凝集させたもの、をそれぞれ作製した。次に、15mLの遠心分離管に、分離液(LymphoPrep:密度1.077±0.001g/mL)を入れて、その上に上記希釈スパイク血又は血球細胞同士を凝集させたものを入れて、800G20分(室温:約23℃)の条件で遠心分離を行った。そして単核球層を分離した。分離した単核球層にリン酸バッファー(PBS)溶液を添加して、遠心分離を再度行い、濃縮を行った。遠心分離後の最下層にできた塊をFBS(ウシ胎児血清)10%添加液体培地(最初の全血量と同じ液量)で懸濁させた。この懸濁液を用いて、ウェルに1mlずつ注入し、37℃で1時間接着させた。その後、PBS溶液で未接着の細胞を洗浄した。次いで、蛍光顕微鏡で接着したがん細胞数をカウントした。
(血球細胞同士の凝集方法)
等量の液体培地を入れて希釈した希釈スパイク血(前記希釈スパイク血を希釈して作製)に、希釈前のスパイク血量の1/20の量の赤血球・白血球凝集抗体試薬であるRosetteSep Human CD45 Depletion Cocktail(STEMCELL Technologies 社製)を、加えて、室温で20分放置して、凝集させた。
11 ウェル
21 親水性ポリマー層
Claims (6)
- 血液又は体液中に含まれる特定細胞捕捉方法であって、
採取した血液又は体液の血球細胞同士を凝集させた後、遠心分離を行い、次いで、親水性ポリマー層に特定細胞を捕捉するもので、
前記方法は、ウェルの内側面の少なくとも一部に親水性ポリマー層が形成されているウェルにより実施され、
前記ウェルが非貫通孔であり、
前記特定細胞は、がん細胞であり、
前記親水性ポリマー層は、ポリ2-メトキシエチルアクリレートで形成されている特定細胞捕捉方法。 - 採取した血液又は体液を希釈した後、前記凝集、前記遠心分離を行う請求項1記載の特定細胞捕捉方法。
- 希釈は、緩衝溶液又は液体培地を用いて行われる請求項2記載の特定細胞捕捉方法。
- 前記血球細胞同士を凝集させる方法が抗原抗体反応を用いる方法である請求項1~3のいずれかに記載の特定細胞捕捉方法。
- 親水性ポリマー層の厚みが10~500nmである請求項1~4のいずれかに記載の特定細胞捕捉方法。
- 請求項1~5のいずれかに記載の特定細胞捕捉方法を用いて捕捉した血液又は体液中の特定細胞を調べる特定細胞検査方法。
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EP3553516B1 (en) | 2023-04-12 |
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US20190250150A1 (en) | 2019-08-15 |
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