Tumor Cell Culture Introduction.doc
1. Repeated post
1. After the cells have grown to a certain amount, the old culture medium is poured out, after digestion with trypsin, Hanks rinses twice, the culture medium without serum is added, and the cell suspension is prepared by pipetting.
2. Take the three culture flasks numbered A, B, and C. First, inoculate the suspension into the A culture flask. After standing still in the incubator for 5 to 20 minutes, gently tilt the culture flask to allow the liquid to concentrate at the corner of the flask and slowly aspirate all the culture fluid, and then inoculate the B culture flask. , Add a little complete culture solution to flask A and place in the incubator to continue the cultivation.
3. After culturing the cells in flask B for 5 to 20 minutes, pour the culture solution into flask C according to the method of treatment A, and then add complete medium to flask B.
2. Mechanical scraping method
1. Marking: Observation under the microscope, using a non-marking pen to grow tumor cells under the back circle of the culture flask.
2. Scraping: Discard the culture solution, extend the sterile glue into the bottle, peep under the naked eye or microscope, and scrape the unmarked space.
3. Rinse once or twice with Hanks solution to remove scraped cells.
4. Inject culture medium to continue culturing. If fibroblasts are still found, they can be scraped repeatedly until completely removed.
3. Digestion and elimination
1. First rinse the medium cells with a mixture of 0.5% trypsin and 0.02% EDTA (1: 1), then change to a new mixture to continue digestion, and peep under an inverted microscope and shake the culture flask from time to time, until half of the cells fall off After that, the digestion stops immediately.
2. Inhale the digestive fluid into the centrifuge tube, centrifuge to remove the supernatant, suck it into another bottle, add the culture solution to the incubator, and add new culture solution to the original bottle to continue the cultivation. After treatment with this method, fibroblasts are easier to shed than tumor cells. After several repeated treatments, fibroblasts may be removed.
4. Collagenase digestion
1. It can be digested with 0.5mg / ml collagenase, and peep under the inverted microscope while digesting. When it is found that the fibroblasts are removed, the digestion is terminated.
2. After washing once with Hanks, replace with new culture medium and continue culturing to obtain pure tumor cells. If the fibroblasts are not removed, they can be repeated again.
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