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            武漢原生原代生物醫藥科技有限公司

            PriCells: Identification and expansion of the tumorigenic lung cancer stem cell population

            時間:2021-11-10 閱讀:230
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            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.

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