Cell line refers to the cell population that reproduces after the first successful passage of primary cell culture, and also refers to cultured cells that can be continuously passaged for a long time. Drug-resistant cell lines are developed by simulating the conditions experienced by cancer patients during chemotherapy. Compared with the parental cell line, drug-resistant cell lines show 2-8 times more resistance. Stable cell lines refer to cell lines that are constructed based on a certain cell line and continuously overexpress or interfere with a specific gene, including knockdown (KD) cell lines, knockout (KO) cell lines, and knockin (KI) cell lines.
I. Construction of drug-resistant cell lines
1. Experimental principle
Long-term contact of cells with low-dose drugs causes changes in the drug chemistry of the cells themselves, and drug resistance-related proteins such as P-glycoprotein appear on the cell membrane, making the cells gradually tolerant to the drugs. Resistant cells multiply in large numbers and produce resistant offspring. As the drug concentration increases, their tolerance gradually increases, thus constructing a cell line that is resistant to specific drugs.
2. Experimental steps
2.1 Take cells in the logarithmic phase and plate them in a 96-well plate at a density of 1×105 cells/well. After overnight culture, detect the half inhibitory concentration (IC50) of the drug;
2.2 Take cells in the logarithmic growth phase (confluence 80%-90%), add drugs with a low initial concentration (recommended to be 1/10-1/5 of the IC50 of the parental cell line) for treatment, and culture them in a 37 ℃, 5% CO2 incubator;
2.3 When the cell density reaches 50%, discard the culture medium, wash twice with PBS, and replace the medium without drugs to continue culturing;
2.4 When the cell growth density recovers to 80%-90% again, repeat the above drug treatment 6-8 times;
2.5 After the cells grow stably at this concentration, increase the drug concentration in sequence and treat them in the same way until the cells can grow stably at the final drug concentration to obtain a drug-resistant cell line;
2.6 Detect the IC50 of the drug-resistant cell line and calculate the resistance index (RI), RI = IC50 of the drug-resistant cell line / IC50 of the parent cell line. If RI>5, it is considered that the drug resistance of the drug-resistant cell line meets the requirements of the drug-resistant strain.
3. Example of experimental results
Under the same drug and drug concentration treatment, the cell survival rate and IC50 of drug-resistant cells HCT116/b-AP15 were higher than those of parental cells HCT116.
II. Construction of KD/KO/KI stable cell lines
1. Experimental principles
The exogenous gene is cloned into a vector with a certain resistance, and the exogenous gene is integrated into the host chromosome by transfecting the host cell. The cell line with the target gene successfully integrated can be obtained by screening with the resistance gene contained in the vector. The lentiviral infection method is currently the most commonly used method for constructing stable cell lines. It has the advantages of efficient integration, efficient transcription, efficient expression, wide host range, high infection efficiency, and integration with cell chromosomes without gene rearrangement. It is an ideal method for preparing stable cell lines.
2. Experimental steps
2.1 Construct the target gene into a suitable lentiviral vector, use 293T cells for lentiviral packaging, and obtain lentivirus containing the target gene;
2.2 Plate cells 24 hours before transfection, and control the cell density at about 80% during transfection;
2.3 Add an appropriate amount of virus suspension to infect the cells the next day, and incubate them in a 37 ℃, 5% CO2 incubator;
2.4 Replace the virus-containing culture medium with fresh culture medium after 24 hours, and continue to culture;
2.5 Start drug addition and screening culture 3-4 days after transfection, until the proportion of fluorescent cells observed under a microscope is 100%, and a stable cell line is obtained.
3. Experimental results example
After the virus carrying the GFP gene infects the cells, a large number of cells expressing fluorescence can be observed under a fluorescence microscope, and a large number of stably transfected cells can be obtained.
References
[1] Tandon N, Thakkar KN, LaGory EL, Liu Y, Giaccia AJ. Generation of Stable Expression Mammalian Cell Lines Using Lentivirus. Bio Protoc. 2018 Nov 5;8(21):e3073.
[2] Milone MC, O'Doherty U. Clinical use of lentiviral vectors. Leukemia. 2018 Jul;32(7):1529-1541.
[3] Maes ME, Colombo G, Schulz R, Siegert S. Targeting microglia with lentivirus and AAV: Recent advances and remaining challenges. Neurosci Lett. 2019 Aug 10;707:134310.
[4] Amaral MVS, DE Sousa Portilho AJ, DA Silva EL, DE Oliveira Sales L, DA Silva Maués JH, DE Moraes MEA, Moreira-Nunes CA. Establishment of Drug-resistant Cell Lines as a Model in Experimental Oncology: A Review. Anticancer Res. 2019 Dec;39(12):6443-6455.
[5] Li HL, Xie SM, Zhang L, Cai CJ, Wang W, Huang J, Wang DY, Wen DP, Deng QH, Zhong NS, He JX. Establishment and characterization of a new drug surviving cell line Am1010, derived directly from muscle metastases of a human lung adenocarcinoma patient with multi-drug-resistance to cisplatin, taxol, and gefitinib. Acta Pharmacol Sin. 2010 May;31(5):601-8.