What to do when encountering fibroblast interference during the extraction of primary tumor cells? There are 5 ways to easily remove it

1. Mechanical scraping method

Made with a rubber scraper at the end of the stainless steel wire (cut into a triangular shape with a rubber stopper and inserted into the stainless steel wire) or wrapped with a small amount of degreased cotton, put it into a test tube for high-pressure steam sterilization and use it as a backup (it can also be scraped off with a special electric cautery). The procedure is as follows:

(1) Mark, observe under a microscope, and use a marker to circle the desired tumor cell location on the back of the culture bottle (dish).

(2) Scrape and discard the culture medium, scrape the sterile glue into the bottle (dish), and under naked eye or microscope observation, scrape off the unmarked cell space.

(3) Wash Hanks solution 1-2 times to remove scraped cells.

(4) Add the culture medium to continue cultivation. If there are still fibroblasts left, repeat scraping until thoroughly scraped.

2. Repeated wall sticking method

Based on the slow adhesion speed of tumor cells compared to fibroblasts, and combined with the use of serum-free nutrient solution, the cell suspension containing two types of cells was repeatedly adhered to the wall, allowing the two types of cells to separate from each other. The method is as follows (same as wall attached generation):

(1) After the cells have grown to a certain amount, pour out the old solution, digest it with 0.25% trypsin, wash twice with Hanks solution, add serum-free culture medium, blow and disperse, and prepare a single cell suspension.

(2) Take three culture bottles numbered A, B, and C. Firstly, inoculate the cell suspension into bottle A and let it sit in a incubator for cultivation for 5-20 minutes. Gently tilt the culture bottle to allow the liquid to concentrate at the corner of the bottle. Slowly suck out all the culture material and inoculate it into bottle B. Then, add complete culture medium to bottle A and continue cultivation in the incubator.

(3) After the cells in bottle B are incubated for 5-20 minutes, the culture medium and cell suspension in bottle B are aspirated and injected into bottle C according to the method used for bottle A. Then, complete culture medium is added to bottle B, and a small amount of calf serum (concentration of 10%) is added to bottle C.

Three bottles were cultured together in the incubator. If the operation was successful and observed the next day, it can be seen that in bottle A, fibroblasts are mainly present, while in bottle B, two types of cells are mixed. In bottle C, epithelial cells (cancer cells) are mainly present. If necessary, they can be repeatedly processed several times until the cancer cells are purified.

3. Digestive exclusion method

(1) Firstly, rinse the cultured cells with a mixture of 0.25% trypsin and 0.02% EDTA (1:1), and then add a new mixture to continue digestion until half of the cells begin to shed, immediately stopping digestion.

(2) Centrifuge the digestive fluid and discard the supernatant. The settled cells are resuspended in culture medium and inhaled into another culture bottle. Incubate in a incubator and add new culture medium to the original bottle for further cultivation. After this method of treatment, considering that fibroblasts shed earlier than tumor cells, the remaining cells in the original bottle are mostly tumor cells. After several repeated treatments, fibroblasts can be removed completely.

4. Collagenase digestion method

This method utilizes the sensitivity of fibroblasts to collagen and selects through digestion.

(1) Collagenase digestion with a concentration of 0.5mg/mL can be used, and digestion can be performed while observing under a microscope. Once fibroblasts are removed, digestion is terminated.

(2) After washing and treating with Hanks solution once, replace with a new culture medium and continue to cultivate, pure tumor cells can be obtained. If not completely cleared, they can be repeated again.

5. Density gradient centrifugation method

A density gradient separation solution with a specific gravity of 1.025-1.085 was prepared using meglumine diatrizoate and sucrose. After adding the cell suspension, it was centrifuged at 2500r/min for 10 minutes. Fibroblasts were identified in layers with a specific gravity of 1.025-1.050, and epithelial cells were identified in layers with a specific gravity of 1.065-1.085. The collected epithelial cells were washed three times and cultured to obtain pure tumor cells. Adding an appropriate amount of SOD during extraction can also inhibit the growth of fibroblasts.

Qida Biological Tumor Cell Ballulation Culture Medium is a serum free, animal component free culture medium that allows cell lines and primary tumor stem cells to form balls; After testing and verification, it has the following four main characteristics:

1. Most tumor cell lines can be directly spheroidized using TSCM

2. Can cultivate complex spheres and organoids

3. Can maintain the passage and amplification of homogeneous tumor balls

4. There is no serum or animal derived protein component, which can maintain the uniformity and verifiability of the experiment.