Tumor cells in vitro subculture and conservation - Clinical Medicine Articles - China Articles

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I. Cell Recovery and Culture

Tumor cells stored in liquid nitrogen or at -80°C are rapidly thawed in a 37°C water bath. The swollen cell suspension is then mixed with 8.0 ml of culture medium, followed by centrifugation at 1500 rpm for 3 minutes. After discarding the supernatant, 8.0 ml of fresh medium is added to resuspend the cell pellet. This process is repeated once more, and finally, the cell pellet is resuspended in 1.0 ml of medium for use.

A new 75 cm² flask is prepared by adding 14.0 ml of culture medium, into which the 1.0 ml of tumor cell suspension is introduced. The cells are then cultured in a 37°C incubator with 5% CO₂. If the cells are suspended, the medium typically turns yellow within 3–4 days, allowing for subculturing of 5.0 ml into a new flask. This process can be repeated across 3–4 flasks, enabling the tumor cells to reach a log phase population of up to 1–1.5 × 10⁷ cells, depending on experimental requirements.

If the cells grow adherently, after 3–4 days, they will form an 80–95% confluent monolayer. At this point, the supernatant is removed, and the cells are treated with 0.5 mM EDTA (or 0.25% trypsin solution, 1.0 ml) for about 3–5 minutes. Under an inverted microscope, when approximately 90% of the cells round up, they are gently detached using a curved pipette and transferred into a 15 ml centrifuge tube. Centrifugation at 1500 rpm for 3 minutes follows, after which the supernatant is discarded. A small amount of medium is added to resuspend the cells, and the culture is expanded into three 75 cm² flasks for further growth.

II. Cell Cryopreservation

Logarithmically growing tumor cells are collected in a 75 cm² flask as described earlier. After centrifugation at 1500 rpm for 3 minutes, the supernatant is discarded, and the cell pellet is resuspended in 3.0 ml of cryopreservation solution (10% DMSO in fetal bovine serum). The suspension is then divided into 2–3 cryovials, labeled with the cell line name, date, and operator’s name. These vials are stored at -80°C overnight and then transferred to liquid nitrogen for long-term storage.

Note: Cells preserved at -80°C can remain viable for up to 6–12 months, while those stored in liquid nitrogen can be kept for 5–10 years or even longer. All equipment used must be sterilized at 121°C for 30 minutes, and the culture medium should be filtered through a 0.22 µm filter. All procedures must strictly follow aseptic techniques to prevent contamination from bacteria, fungi, viruses, or other pathogens.