Method for culturing lung cancer stem cells under 3D culture conditions

Abstract

The invention discloses a method for culturing lung cancer stem cells under 3D culture conditions and belongs to the field of cell culture methods. The method comprises the following steps of placing human lung adenocarcinoma cell line A549 in an incubator for culturing with a RPMI-1640 complete culture medium containing 10% fetal calf serum, taking a cell suspension in a logarithm growth period, adjusting the concentration of the cells to 5*10<4>cells / ml and adding 200 mu L / well into a 96-well plate in which 50 mu L / well BME is spread; on the third day, changing the culture medium for the A549 cells with a RPMI-1640 complete culture medium containing IGF-1 and FGF and further culturing; digesting BME through a proteolytic enzyme, recovering the A549 cells from a recovery liquid and finally identifying the cells. By such design, the A549 cells form cloned mass similar to in-vivo tumor mass in vitro, which is closer to the three-dimensional growth state in the human body and thus the stable separation and effective amplification of stem cells are achieved. The cancer stem cells capable of resisting anticancer drugs are screened.

Description

technical field [0001] The invention relates to a conditional 3D cell culture method, which is a method capable of efficiently separating and expanding lung cancer stem cells, and can be used to establish an in vitro screening system for innovative tumor drugs. technical background [0002] Lung cancer is a malignant tumor with the highest morbidity and mortality rate in the world, which seriously threatens human health. Among them, non-small cell lung cancer (NSCLC) accounts for about 80%-85% of lung cancer. In recent years, with the advancement of surgical techniques and the promotion of standardized radiotherapy and chemotherapy regimens, the clinical treatment level of NSCLC has been greatly improved, but the 5-year survival rate of NSCLC has not been effectively improved. The reason is that in addition to the malignant characteristics of NSCLC, which are highly invasive and prone to recurrence, the limitation of clinical drug treatment is also an important factor restri...

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problem that the number of tumor stem cells in tumor cells is extremely small, and most of them are in the quiescent phase. It is difficult to effectively separate and amplify in vitro by using traditional 2D cell culture, so it is also difficult to establish a tumor cell that is suitable for innovative drug screening. To address the problem of in vitro pharmacological models of cancer stem cells, and provide a conditional 3D culture method for efficiently isolating and expanding lung cancer stem cells

Images