I. Overview of tumor cell culture
The primary culture of tumor cells is substantially similar to the conditions of primary culture of normal cells. Generally, the primary culture medium for tumor cells, 5 to 10% serum concentration, cell culture additives, antibiotics, and the like can be used. Or in the primary culture, it is necessary to add a special additive for primary cell culture, or the original patient serum (1~2%) to facilitate cell growth. Cell growth factors such as insulin, hydrocortisone, estrogen, and the like are used. Animal vector culture can also be considered.
Second, the top ten biological characteristics of tumor cells
Self-Sufficiency in Growth Signals;
Insensitivity to Antigrowth Signals;
Resisting Cell Death;
Limitless Replicative Potential;
Sustained Angiogenesis;
Tissue Invasion and Metastasis;
Avoiding Immune Destruction;
Promotes tumor inflammation (Tumor Promotion Inflammation);
Degegulating Cellular Energetics;
Genome Instability and Mutation;
Third, the basic method of tumor cell isolation and culture
Different growth promoting factors are selected depending on the cell type. The methods for primary isolation of tumor tissue into tumor cells include tissue block method, enzyme digestion method, sputum net method, and exfoliated cell method.
1. The tissue block method removes the fat, connective tissue and necrotic part of the obtained tumor tissue, washes it with Hanks solution 3 times in the dish, cuts the tissue, cuts into small pieces of 1~2mm3, and inoculates in the culture bottle (or dish). , 370C, 5% CO2 or capped and stuffed in a common incubator for cultivation.
2, enzymatic digestion in the shredded tissue, cut into 1 ~ 2mm3 small pieces, add 0.25% trypsin or 2000U / ml collagenase, digested in 370C water bath for 30min or longer, remove the digestive juice, wash with lotion After 3 times, the medium was washed once, suspended in complete medium, and pipetted with a pipette to prepare a cell suspension, and counted. At a concentration of (5~10) x 108 cells/L, cultured in a professional primary tumor cell complete culture system, 370C, 5% CO2 in a vial (or dish).
3, é’½ 法 在 在 ä¸€å¼ ä¸€å¼ ä¸€å¼ ä¸€å¼ ä¸€å¼ ä¸€å¼ ä¸€å¼ ä¸€å¼ ä¸€å¼ ä¸€å¼ åœ¨ 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在Put it into the culture dish, make the tissue block under the net tightly contact with the dish wall. In the complete culture system of professional primary tumor cells, culture at 370C, 5% CO2, and change the liquid every 2~3 days. After 5 days, the cells were removed from the network and a pure monolayer of tumor cells was formed at the site equivalent to the tumor tissue.
4. Exfoliated cell method After removing the fat and connective tissue from the fresh tumor tissue, the washing liquid is washed twice, and the tumor tissue is cut into thin slices with a blade. At the same time, many tumor cells are detached, and the exfoliated cells are washed. By adding complete medium, the pure upper layer cells can be obtained in a professional primary tumor cell complete culture system, cultured in a culture flask (or dish), 370C, 5% CO2, and changed every 2-3 days. Once, the tumor cells gradually grow into a single layer in 7-10 days.
remember:
1. The whole process and surrounding links must be strictly sterile. The pollution may be accompanied by a certain link or whole link of primary cell separation, culture, cryopreservation, resuscitation, transportation, etc. Pollution must be the number one problem of primary cell culture failure. .
2, must use professional, supporting primary cell complete culture system. The primary cell culture system for primary cell isolation and primary generation primary cell culture is the best primary cell culture system for primary cell culture of this variety.
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