This article details a method for generating chemoresistant prostate cancer cells through a series of drug treatments. The process involves exposing sensitive cells to escalating doses of docetaxel, allowing for the selection of resistant clones.
To generate chemoresistant cells, begin by taking human prostate cancer cell suspension in a culture flask. Incubate the flask to facilitate cell adherence and their division to form a monolayer.
Supplement the cells with an anti-cancer drug solution at an optimum concentration and incubate. The drug molecules enter the cells and induce apoptosis in sensitive cells.
Some cells express special drug efflux transporters or pumps that periodically remove the drugs. This mechanism prevents drug accumulation, enabling these cells to withstand the effect of anti-cancer drugs and become chemoresistant.
Aspirate the drug- and debris-containing media from the culture. Supplement with fresh drug-free growth media to support the growth of chemoresistant cells. Discard the spent media. Trypsinize the cells to detach them from the culture flask and transfer them to a tube.
Centrifuge the tube to pelletize the cells. Remove the trypsin-containing supernatant. Resuspend the cells in fresh growth media. Grow the cells in a new culture flask.
Subsequently, repeat the drug treatment and cell culture steps to expose the chemoresistant cells to escalating doses of the same anti-cancer drug. This technique screens the culture by eliminating all the sensitive cells to generate a pool of highly chemoresistant surviving cells.
To begin this procedure, plate DU145 or 22Rv1 cells in the 150-centimeter squared flasks containing 20 milliliters of media. After 24 hours, when the cells are at about 70% to 80% confluence, add docetaxel at 5 nanomolar.
After 72 hours, aspirate the drug-containing media and add fresh docetaxel-free media. Change the media every 3 to 4 days, and wait for 1 to 2 weeks until clones appear in the flask. Aspirate the media. Carefully wash the cells with 15 milliliters of PBS and incubate them with 4 milliliters of 0.05% trypsin-EDTA for 3 to 5 minutes at 37 degrees Celsius to detach the cells from the flask surface. Then, resuspend the trypsinized cells from the flask using 8 milliliters of fresh media.
Pool the cells from all treated flasks and pellet them by centrifugation. Following that, remove the supernatant. Resuspend the cell pellet in 20 milliliters of fresh media and plate the cells in 150-centimeter squared flasks.
After 24 hours, when the cells are at about 70% to 80% confluence, add 5 nanomolar docetaxel again. Repeat the procedures as shown previously until the clones appear in the flask. Then, plate the cells in 150-centimeter squared flasks again.
After 24 hours, when the cells are at about 70% to 80% confluence, treat them with 10 nanomolar docetaxel. Repeat the same steps in a docetaxel dose-escalating manner and keep pooling the surviving clones after every concentration treatment. At the end of the process, you will obtain a pool of resistant cells ready for experimental use.
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