Primary Tumor Extraction and Culture Protocol

Primary culture of tumor cells is a common experimental method, which is used to study the growth, differentiation, metastasis and other biological characteristics of tumor cells. Primary culture refers to the first generation of cells isolated from tumor tissue that are cultured in vitro and have similar biological characteristics to the original tumor tissue. It is an important tool for studying tumor biology.

一、Preparation of reagents:

Phosphate buffered saline (PBS) (Qida article No.: SD0029), Fetal bovine serum (to be inactivated) (Qida article No.: SD0200), trypsin(Qida article No.: SD0003), Hepes (Qida article No.: SD0009), MEM basic culture medium (Qida article No.: MD0300), B-27 cell additive (Qida article No.: SD0011), PSN antibiotic (Qida article No.: SD0034), polylysine (Qida article No.: SD0042)

二、 Steps for extracting tumor cells:

1. Perform in vitro total resection of the tumor, remove blood, water, and capsule on qualitative filter paper, and use ophthalmic scissors to shred the tissue in a small amount of PBS.

2. In an oscillating bath at 37 ° C, treat the chopped tissue with 11 ml Digestive enzyme of each sample (in MEM containing 20mM HEPES, 1:1000 Paint thinner of trypsin 2.5%) for 30 minutes. After digestion, the separated cells were filtered through a 70 µ m sieve.

3. Add 5 mL of 10% heat inactivated FBS to inactivate trypsin. Centrifuge cells at 450 x g for 10 minutes, then resuspend in MEM containing 20mM HEPES and 10% FBS.

4. Centrifuge the cells at 300X g for 20 minutes, then resuspend in the culture medium.

Spread tumor cells onto a 6-well tissue culture plate coated with poly L-lysine (0.1mg/ml concentration) at a concentration of 2 million cells per well.

5. Use corresponding culture medium with 10% FBS, 1% B-27, and 1% PSN to culture cells (DMEM high glucose and McCoy's 5A based primary tumor cell culture are the most common), and add growth factors according to cell growth characteristics

6. Collect the tumor cell conditioned medium from the fusion culture of the tumor cell line maintained in the basic medium for 48 hours.

三、 Pay attention to the details, primary tumor cell culture is as simple as this:

(1) The culture material should be taken from the concentrated and active part of tumor cells, and should be immediately cultured and stored in the culture medium.

(2) During the sampling and cultivation process, it is necessary to prevent bacterial and mold contamination.

(3) Fibroblasts mixed in the culture should be removed as soon as possible.

(4) If there is lymphocyte infiltration in cancer tissue, gradient centrifugation should be used to remove the lymphocytes, otherwise the cancer cells will be killed.

(5) The experiment should prevent cross contamination of other cell lines.

(6) When cancer cells do not grow to a sufficient amount, they should be patient, insist on changing the fluid, and not rush to passage.

(7) Cancer cells often overlap and grow. If there are mixed fibroblasts, digestion and repeated adhesion methods should be used to eliminate them during passage, and cancer cells should be isolated for passage and culture.

(8) Cell growth and reproduction are extremely unstable before reaching the 10th generation, so it is important to be careful when subculturing. When it is necessary to co culture, it is important to avoid subculture. To determine the suitable culture method for the cell.

(9) It is necessary to increase the inoculation concentration appropriately during early passage.

(10) Eliminating fibroblasts is always an important condition in cancer cell culture, and once discovered, it should be eliminated as soon as possible according to the methods introduced above.

Reference:

1. Canoll, P. D., Musacchio, J. M., Hardy, R., Reynolds, R., Marchionni, M. A. and Salzer, J. L. (1996). GGF/neuregulin is a neuronal signal that promotes the proliferation and survival and inhibits the differentiation of oligodendrocyte progenitors. Neuron 17(2): 229-243.

2. Gensert, J. M. and Goldman, J. E. (2001). Heterogeneity of cycling glial progenitors in the adult mammalian cortex and white matter. J Neurobiol 48(2): 75-86.

3. Lei, L., Sonabend, A. M., Guarnieri, P., Soderquist, C., Ludwig, T., Rosenfeld, S., Bruce, J. N. and Canoll, P. (2011). Glioblastoma models reveal the connection between adult glial progenitors and the proneural phenotype. PLoS One 6(5): e20041.