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新聞中心

細胞凍存方法主要操作步驟

發布時間:2022-07-20瀏覽次數:46返回列表

  細胞凍存方法主要操作步驟為:

1、選擇處于對數生長期的細胞,在凍存前一天*換液。將多個培養瓶中的細胞培養液去掉,用0.25% 胰蛋白酶消化。適時去掉胰蛋白酶,加入少量新培養液。用吸管吸取培養液反復吹打瓶壁上的細胞,使其成為均勻分散的細胞懸液。懸浮生產細胞則不要消化處理。然后將細胞收集于離心管中離心(1000r/min,10分鐘)。

2、去上清液,加入含20%小牛血清的完全培養基,于4℃預冷15分鐘后,逐滴加入已無菌的DMSO或甘油,用吸管輕輕吹打使細胞均勻,細胞濃度為3×1061×107/mL之間。

3、將上述細胞分裝于安瓿或冷凍塑料管中,安瓿裝11.5mL在火焰噴燈上封口,封口處要完全封閉,圓滑無勾。冷凍管要將蓋子蓋緊,并標記好細胞名稱和凍存日期,同時作好登記(日期、細胞種類及代次、凍存支數)。

4、將裝好細胞的安瓿或凍存管裝入沙布袋內;置于液氮容器頸口處存放過夜,次日轉入液氮中。采用控制降溫速度的方法也可采用下列步驟:先將安瓿置入4℃冰箱中23小時,再移至冰箱冷凍室內34小時(此步可省略),再吊入液氮容器頸氣態部分存放2小時,蕞后沉入液氮中。

細胞凍存在液氮中可以長期保存,但為妥善起見,凍存半年后,*取出一只安瓿細胞復蘇培養,觀察生長情況,然后再繼續凍存。

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