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I. Cell recovery and culture
The tumor cells preserved in liquid nitrogen or -80 ° C were quickly dissolved in a water bath at 37 ° C, and the swollen tumor cell suspension was mixed with 8.0 ml of the medium, and centrifuged at 1500 rpm for 3 minutes. Discard the supernatant, then pipet 8.0 ml of culture medium to mix the cell pellet, centrifuge at 1500 rpm, centrifuge for 3 minutes, discard the supernatant, and mix the cell pellet with 1.0 ml of medium for use. Another 75 cm square flask was added, and 14.0 ml of the culture substrate was added. The tumor cell-containing suspension (1.0 ml) prepared above was added to the square flask, and cultured at 37 ° C in a 5% CO 2 incubator.
If the tumor cells are suspended, the cell matrix will turn yellow in about 3-4 days, and 5.0 ml of the cell suspension can be subcultured in a square flask. It can be cultured in 3-4 square flasks, and the tumor cells growing in logarithm in a square bottle. Up to 1-1.5 × 107, depending on the test, the number of passages can be determined. If the tumor cells grow adherently, after 3-4 days, the tumor cells grow to 80%-95% monolayer, discard the supernatant, and use 0.5 mM EDTA (indigestible tumor cells with 0.25% trypsin 1.0 ml) ), treat the tumor cells for about 3-5 minutes and observe with an inverted microscope. When 90% of the tumor cells are rounded, they can be blown with a curved tube and the digested cells can be transferred to a 15 ml centrifuge tube at 1500 rpm. Centrifuge for 3 minutes, discard the supernatant, add a little medium to mix, and pass three 75cm2 square bottles to expand the culture.
2. Cell cryopreservation
The logarithmically grown tumor cells were collected in a 75 cm2 square bottle as described above, and centrifuged at 1500 rpm for 3 minutes. The supernatant was discarded and mixed with 3.0 ml of a stock solution (10% DMSO in calf serum). Add to 2-3 preservation tubes, write the name of the tumor cells, time, name of the breeder, store at -80 °C, transfer them to liquid nitrogen the next day (Note: Save at -80 °C Cells for half a year to a year, liquid nitrogen can hold cells for 5-10 years, or even longer). All the above items are subject to high temperature sterilization (121 ° C, 30 minutes), and the culture medium is sterilized by filtration (0.22 uM). All operations must follow the aseptic technique to avoid contamination by bacteria, fungi, pathogens, chlamydia and so on.
Cell recovery and cryopreservation method
Cell recovery and cryopreservation method