I. Cell Recovery and Culture
Tumor cells stored in liquid nitrogen or at -80°C were quickly thawed in a 37°C water bath. After thawing, the swollen cell suspension was mixed with 8.0 ml of culture medium and centrifuged at 1500 rpm for 3 minutes. The supernatant was discarded, and the cell pellet was resuspended in 8.0 ml of fresh culture medium. Another centrifugation at 1500 rpm for 3 minutes was performed, followed by discarding the supernatant again. The resulting cell pellet was then suspended in 1.0 ml of culture medium for further use. Next, a 75 cm² flask was prepared by adding 14.0 ml of culture medium, into which the 1.0 ml cell suspension was introduced. The cells were then incubated at 37°C in a 5% CO₂ incubator.
If the tumor cells are in suspension, the medium typically turns yellow within 3-4 days. At this point, 5.0 ml of the cell suspension can be subcultured into a new 75 cm² flask. This process can be repeated across 3-4 flasks, allowing the tumor cells to grow logarithmically until they reach a concentration of approximately 1-1.5 × 10ⷠcells, depending on the experimental requirements. The number of passages is determined based on the growth rate and the needs of the study.
In cases where the tumor cells adhere to the surface, after 3-4 days, they usually form an 80%-95% confluent monolayer. The medium was then removed, and the cells were treated with 0.5 mM EDTA (in combination with 0.25% trypsin, 1.0 ml total) for about 3-5 minutes while being observed under an inverted microscope. When approximately 90% of the cells had rounded up, they were gently detached using a curved pipette and transferred into a 15 ml centrifuge tube. The cells were centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and the pellet was resuspended in a small amount of medium. These cells were then distributed into three 75 cm² flasks to expand the culture.
2. Cell Cryopreservation
Logarithmically growing tumor cells were collected from a 75 cm² flask as previously described. They were centrifuged at 1500 rpm for 3 minutes, the supernatant was removed, and the cell pellet was resuspended in 3.0 ml of cryopreservation solution (10% DMSO in fetal bovine serum). The cell suspension was then aliquoted into 2-3 cryovials. Each vial was labeled with the cell line name, date, and researcher's name before being placed at -80°C overnight. The next day, the vials were transferred to liquid nitrogen for long-term storage. It is important to note that cells stored at -80°C can remain viable for up to one year, while those kept in liquid nitrogen may last 5-10 years or even longer.
All reagents and equipment used in the procedure were sterilized using high-temperature autoclaving (121°C for 30 minutes), and the culture medium was filtered through a 0.22 µm filter to ensure sterility. All procedures were carried out under strict aseptic conditions to prevent contamination by bacteria, fungi, mycoplasma, and other pathogens. Maintaining a clean and controlled environment is essential for successful cell culture and long-term cell preservation.
Ambulance Car,Emergency Ambulance Car,Electric Ambulance Car,Rescue Ambulance Car
CLW GROUP TRUCK , https://www.clwgrouptruck.net