The cells are sequentially placed in a series of concentrations of MTX for several weeks, with periodic changes to fresh medium containing the same drug concentration. Flasks in which only a small percentage of cells survived and formed colonies were selected for further incubation. These selected cells are further cultured in MTX at the original concentration or 2 to 10 times higher until the desired resistance is achieved.
Operation method
Scheme 14.9 Methotrexate resistance and DHFR amplification
Principle
Sequential exposure of cells to progressively increasing concentrations of a folate antagonist, such as methotrexate (MTX ), results over time in the development of resistance to the toxicity of the drug [ Biedler et al, 1972 ], as a result of amplification of the DHFR gene, and this resistance is usually produced very rapidly, although there may of course be other mechanisms partially or wholly involved in the development of the resistance phenotype, such as, for example, alterations in the transport of folate and/or mutations affecting the structure or affinity of the enzyme. alterations in folate transport and/or mutations affecting enzyme structure or affinity.
Materials and Instruments
Chinese hamster cells or fast-growing human or mouse cell lines Amethopterin sodium salt Move 1. Cloning of cultured parental cells to form a population of vigorously growing, genotypically homogeneous cells for screening. For more product details, please visit Aladdin Scientific website.
0.15mol LNaCl
Tissue culture flasks Straws Thymidine and hypoxanthine free flasks Inverted microscope Liquid nitrogen tanks
2. Dilute MTX with sterile NaCl 0.15 mol/L ( 0.85 % ), clinically packaged at a concentration of 2.5 mg/ml.
3. 2. 5×105 cells were planted in several identical culture flasks, and complete medium without MTX or with 0.01 μg/ml, 0.02 μg/ml, 0.05 μg/ml and 0.1 μg/ml MTX was added to each culture flask, and the pH of the medium was adjusted to 7.4, and the cells were incubated at 37°C for 5~7 days.
4. Observe the cells under an inverted microscope, if a small portion of the cells in the culture flask appear to be growing clonally, and some of the remaining cells are enlarged and attached to the stroma layer and may be dying, select the cells in such flasks and replace them with fresh medium containing the same amount of MTX to continue the culture.
5. If necessary, change the fresh medium and incubate for another 5~7 days, but the cells must always be in the MTX environment. When the cell density reaches 2~10×106 cells/flask, pass the cells into new culture flasks at 2. 5×105 cells per flask, and add the original concentration of MTX and 2~10 times the original concentration of MTX, respectively.
6. 5~7 days later, observe the new cells and the cells with higher concentration of MTX, change the medium and select the usable cells, the method is the same as before.
7. Cells from each passaging step are continuously screened with progressively higher drug concentrations until the required level of resistance is obtained. It takes 2 to 3 months to obtain low to moderate levels of resistance, increased DHFR activity, and/or altered transporter capacity in Chinese homozygous hamster cells; for Chinese hamster cells, mouse cells, or fast-growing human cells, it can take 4 to 6 months or longer to obtain high levels of resistance and enzyme overproduction in the mutant strains.
8. Periodically freeze the screened cells in liquid nitrogen.