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    • 3. 发明公开
    • 폐암세포의 분리 및 부유배양 기법을 이용한 증식
    • 使用悬浮培养方法分离肺癌细胞及其增殖
    • KR1020140047343A
    • 2014-04-22
    • KR1020120113492
    • 2012-10-12
    • (주)차바이오텍
    • 서민석박순정문성환정형민
    • C12N5/09C12Q1/24C12Q1/25G01N33/15
    • The present invention relates to culture conditions for effective isolation and proliferation of lung cancer cells from a lung cancer tissue of a patient, and a method for screening a patient-specific cancer therapeutic agent using the lung cancer cells cultured thereby, and more specifically, to a method for isolating and culturing lung cancer cells from a lung cancer tissue and a method for screening a patient-specific cancer therapeutic agent using the lung cancer cells prepared by the method. The method for isolating and culturing lung cancer cells from a lung cancer tissue, comprises the steps of: 1) cutting a lung cancer tissue isolated from a patient into a size of 40-60 μm in diameter; 2) allowing the cut tissue in step 1) to react with an enzyme to be separated into single cells; 3) proliferating the separated single cells in step 2) in a serum-free ACL4 medium or N2 added medium on a low-adhesion culture dish through suspension culture; and 4) subculturing the cells, which proliferate through the suspension culture in step 3), using Accutase, wherein step 4) does not require a separate Accutase reaction termination step. [Reference numerals] (A) Isolating cells from a cancer tissue according to the conventional art; (AA) Solid tumor; (B) Isolating and culturing lung cancer cells from a lung cancer tissue according to the present invention; (BB) Collagenase degradation; (CC) Single cell suspension; (DD) Cells in agar matrix; (EE) Incubation for 6-8 days; (FF) Cell population formation; (GG) Clonogenic cell isolation; (HH) Isolated clonogenic cell re-dispensing; (II) Cutting into a size of 40-100 um; (JJ) Suspension culture in an ultra-low adhesion (or pluronic F127 coating) culture dish; (KK) Effective cell Formation and culture in a serum-free medium; (LL) Suspension-cultured cell subculture using Accutase; (MM) Adherent culture; (NN) Suspension culture; (OO) EPCAM expression (%); (PP) Adherent culture; (QQ) Suspension culture; (RR) Culture and proliferation through suspension culture for suppressing an EMT phenomenon occurring due to adherent culture
    • 本发明涉及用于从患者的肺癌组织中有效分离和增殖肺癌细胞的培养条件,以及使用由其培养的肺癌细胞筛选患者特异性癌症治疗剂的方法,更具体地,涉及 用于从肺癌组织分离和培养肺癌细胞的方法和使用通过该方法制备的肺癌细胞筛选患者特异性癌症治疗剂的方法。 从肺癌组织分离和培养肺癌细胞的方法包括以下步骤:1)将从患者分离的肺癌组织切成直径为40-60μm的大小; 2)允许步骤1)中的切割组织与待分离成单细胞的酶反应; 3)通过悬浮培养在低粘附培养皿中的无血清ACL4培养基或N 2添加培养基中的步骤2)中分离的单细胞增殖; 和4)使用Accutase,通过步骤3)中的悬浮培养物传代培养细胞,其中步骤4)不需要单独的Accutase反应终止步骤。 (附图标记)(A)根据现有技术从癌组织分离细胞; (AA)实体瘤; (B)根据本发明从肺癌组织中分离和培养肺癌细胞; (BB)胶原酶降解; (CC)单细胞悬液; (DD)琼脂中的细胞; (EE)孵育6-8天; (FF)细胞群体形成; (GG)克隆细胞分离; (HH)分离的克隆细胞重新分配; (二)切成40-100um的尺寸; (JJ)在超低粘附(或pluronic F127涂层)培养皿中的悬浮培养; (KK)有效细胞在无血清培养基中形成和培养; (LL)使用Accutase的悬浮培养细胞传代培养; (MM)贴壁培养; (NN)悬浮培养; (OO)EPCAM表达(%); (PP)粘附培养; (QQ)暂停文化; (RR)通过悬浮培养培养和增殖以抑制由粘附培养引起的EMT现象