JP6518985B2 - がん細胞捕捉方法 - Google Patents
がん細胞捕捉方法 Download PDFInfo
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Description
前記たんぱく質濃度を減少させる方法は、採取した体液に遠心分離を施して上澄みを除去するものであることが好ましい。
前記親水性ポリマー層の厚みは、2〜200nmであることが好ましい。
本発明のがん細胞捕捉方法は、例えば、図1の医療用検査装置1(マルチウェルプレート1)を用いて実施できる。
(1)、(2)の保持時間は、ウェル11の大きさ、導入する液種、等により適宜設定すれば良いが、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。保持後、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2.5W/V%メタノール溶液を作製した。
市販PMMA製プレート内に、作製したポリ2−メトキシエチルアクリレート溶液(2.5W/V%)を注入し、30分室温放置した後、液をピペットで吸出し、乾燥することで、図1の親水性ポリマー層が形成されたマルチウェルプレートを有する検査装置を作製した。
PBS溶液(リン酸緩衝生理食塩水)に線維肉腫(HT−1080)を懸濁させて、細胞数を血球計測管でカウントした後、PBS溶液内の細胞濃度がある値A1になるように更にPBS溶液を加えて、細胞濃度を調整した。次に、播種密度(濃度)が2850cells/cm2に計算上なるように血液を混濁させた。この場合、血液と等量のPBS溶液を加えたことになった。
PBS溶液(リン酸緩衝生理食塩水)に線維肉腫(HT−1080)を懸濁させて、細胞数を血球計測管でカウントした後、PBS溶液内の細胞濃度がある値A2になるように更にPBS溶液を加えて、細胞濃度を調整した。次に、播種密度(濃度)が2850cells/cm2に計算上なるように血液を混濁させた。この場合、血液と血液の2倍量のPBS溶液を加えたことになった。
DMEM(液体培地)に線維肉腫(HT−1080)を懸濁させて、細胞数を血球計測管でカウントした後、DMEM内の細胞濃度がある値A1になるように更にDMEMを加えて、細胞濃度を調整した。次に、播種密度(濃度)が2850cells/cm2に計算上なるように血液を混濁させた。この場合、血液と等量のDMEMを加えたことになった。
DMEM(液体培地)に線維肉腫(HT−1080)を懸濁させて、細胞数を血球計測管でカウントした後、DMEM内の細胞濃度がある値A2になるように更にDMEMを加えて、細胞濃度を調整した。次に、播種密度(濃度)が2850cells/cm2に計算上なるように血液を混濁させた。この場合、血液と血液の2倍量のDMEMを加えたことになった。
線維肉腫(HT−1080)を剥離液を用いて懸濁させ、一部をPBS溶液に混濁させて、細胞数を血球計測管でカウントした後、この値を用いて、血液に線維肉腫(HT−1080)の入った剥離液を混濁させた。このとき、播種密度(濃度)が2850cells/cm2に計算上なるように血液を混濁させた。次に、混濁させた血液に遠心分離(3000rpm、10分)を行い、上澄みを捨てて、残った血液を取り出した。
線維肉腫(HT−1080)を剥離液を用いて懸濁させ、一部をPBS溶液に混濁させて、細胞数を血球計測管でカウントした後、この値を用いて、血液に線維肉腫(HT−1080)の入った剥離液を混濁させた。このとき、播種密度(濃度)が2850cells/cm2に計算上なるように血液を混濁させた。次に、混濁させた血液を遠心分離(3000rpm、5分)を行い、上澄みを捨てて、残った血液を取り出した。
線維肉腫(HT−1080)を剥離液を用いて懸濁させ、一部をPBS溶液に混濁させて、細胞数を血球計測管でカウントした後、この値を用いて、血液に線維肉腫(HT−1080)の入った剥離液を混濁させた。このとき、播種密度(濃度)が2850cells/cm2に計算上なるように血液を混濁させた。次に、混濁させた血液を遠心分離(1500rpm、10分)を行い、上澄みを捨てて、残った血液を取り出した。
線維肉腫(HT−1080)を剥離液を用いて懸濁させ、一部をPBS溶液に混濁させて、細胞数を血球計測管でカウントした後、この値を用いて、血液に線維肉腫(HT−1080)の入った剥離液を混濁させた。このとき、播種密度(濃度)が2850cells/cm2に計算上なるように血液を混濁させた。
ウェル内面の親水性ポリマー層の膜厚は、TEMを使用し、加速電圧15kV、1000倍で測定(撮影)した。
各検査試料(がん細胞含有血液)をウェルに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、免疫染色を行い、蛍光顕微鏡で接着したがん細胞数をカウントトした。なお、比較例1の接着数を1.0として、相対値で比較した。
11 ウェル
21 親水性ポリマー層
Claims (5)
- たんぱく質濃度を減少させる方法が、採取した体液を希釈するものである請求項1記載のがん細胞捕捉方法。
- 希釈が緩衝溶液又は液体培地を用いて行われる請求項2記載のがん細胞捕捉方法。
- たんぱく質濃度を減少させる方法が、採取した体液に遠心分離を施して上澄みを除去するものである請求項1記載のがん細胞捕捉方法。
- 体液が血液である請求項1〜4のいずれか記載のがん細胞捕捉方法。
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