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PriCells: Identification and expansion of the tumorigenic lung cancer stem cell population
PriCells: Identification and expansion of the tumorigenic lung cancer stem cell population
Lung cancer contains a rare population of CD133+ cancer stem-like cells able to self-renew and generates an unlimited progeny of non-tumorigenic cells. The current technology allows the establishment of long-term lung cancer stem cell cultures from about one-third of the tumors.
1. Tumor samples were obtained.
2. Surgical specimens were washed several times and left overnight in culture system.
DMEM–F12 medium supplemented
high doses of penicillin/streptomycin
amphotericin B
3. Tissue dissociation was carried out by enzymatic digestion (20?μg/ml collagenase II) for 2?h at 37°C.
4. Recovered cells were cultured at clonal density in serum-free medium.
50?μg/ml insulin
100?μg/ml apo-transferrin
10?μg/ml putrescine
0.03?mM sodium selenite
2?μM progesterone
0.6% glucose
5?mM HEPES
0.1% sodium bicarbonate
0.4% BSA
glutamine and antibiotics
20?μg/ml EGF
10?μg/ml bFGF.
5. Flasks non-treated for tissue culture were used to reduce cell adherence and support growth as undifferentiated tumor spheres.
6. The medium was replaced or supplemented with fresh growth factors twice a week until cells started to grow forming floating aggregates.
7. Cultures were expanded by mechanical dissociation of spheres, followed by re-plating of both single cells and residual small aggregates in complete fresh medium.
8. Stem cell medium was replaced with Bronchial Epithelial Cell Growth Medium in tissue culture-treated flasks.
9. Identification: Antibodies used were PE-conjugated anti-CD133/1, PE-conjugated anti-CD133/2 or APC-conjugated anti-CD133/1, anti-CD56/N-CAM, FITC-conjugated anti-epithelial membrane antigen, anti-human CKs, anti-CEA, FITC-conjugated anti-CD34 and anti-CD45, anti-CD31.
10. Single-cell suspensions from lung cancer specimens were prepared.
11. After thawing, cells were double stained with PE-conjugated anti-CD133/1 antibody and FITC-conjugated anti-Ep-CAM antibody.
12. Purity of the CD133+ and CD133? cell populations was evaluated.