From A549 and H1299 cells, After isolation which media can be used to maintain cancer stem cells?
Hi Akash,
I have worked in lung cancer cell lines. I hope the following will be useful for your research.
For isolating the lung cancer cells: 1.Fresh lung cancer specimens were should be obtained and were cut into 0.5-mm sections following the removal of visible blood vessels and necrotic tissue.
2.The tissue specimens were washed numerous times with D-Hank’s solution and left overnight in DMEM with Ham’s nutrient mixture F-12 (DMEM-F12) supplemented with high doses of penicillin/streptomycin and amphotericin B to avoid contamination.
3.The specimens were enzymatically digested in 50 ml BD Falcon (supplemented with collagenase IV (final concentration, 0.1%) and hyaluronidase (final concentration, 0.1%) for 1 h under 5% CO2 at 37°C.
4.The remaining cell debris was removed by passing the cells through a 70μm-diameter disposable cell mesh filter and centrifuging for 15 min at a speed of 400 × g.
For culturing A549 cell lines: RPMI-1640 medium (Gibco, Thermo Scientific) with 10 % fetal bovine serum (FBS) (Gibco) and 1 % penicillin–streptomycin. Use 0.25 % trypsin–EDTA (Gibco) as the cell dissociation reagent. Incubate both cells in a humidified incubator at 37 C supplied with 5 % carbon dioxide. Maintain the cells routinely in 75 cm2 tissue culture flasks and harvest by trypsin treatment when the cells are in the logarithmic phase of growth or at 80 % confluence for flow cytometry analysis.
(1. The prepared growth medium needs to be filtered using a 0.22 μM syringe filter to prevent microbial growth in the cell culture. 2. pH stability of the cell culture needs to be maintained throughout the entire process to ensure successful cell cultivation. pH can be affected by several parameters, including CO2 concentration. Therefore, it is crucial for cell cultivation to be performed in a CO2 incubator. Using 5 % CO2 helps maintain the optimum pH for cell cultivation. Changes of the pH value might have negative consequences on cell growth. 3. Differentiation medium should be changed every third day to prevent drastic changes in the pH of the culture due to metabolic waste accumulation.)
Hope it will be useful for you.
Thanks.
Hope these articles will help you.
Article Lung cancer stem cells—characteristics, phenotype
Article Nonadhesive Culture System as a Model of Rapid Sphere Format...
Dear Bargavi Purushothaman , I am looking for the isolation of CSCs from A549 cell line
Dear Akash,
I feel that instead of confusing with word "isolation", if we use "enrichment of CSCs from cancer cells" it will give better insight. we are also working on CSCs from breast cancer cells where we enrich CSCs like cells from breast cancer cell lines using serum free media containing specific growth factor. its a kind of sphere culture/spheroids (3D)/tumorosphere since CSCs are small population and resides within tumors.
Dear Saurabh,
Thanks for correcting me, yeah its enrichment what I am talking about. Could you please send me detail protocol. It would be of great help.
Dear Akash,
I am attaching few articles where Lung cancer stem cells culturing is nicely described. You can follow them and see. Since in our lab we works with breast cancer stem like cells and Glioma stem cells, in which three major growth factor plays role in sphere culture i.e., B27, b-FGF, and EGF. I found similar component were used to culture Lung CSCs. Even I have attached our lab published paper also. Hope it will help you.
Lung CSCs
Article Identification and Characterization of Cells with Cancer Ste...
Article Cancer Stem Cells in Small Cell Lung Cancer Cell Line H446: ...
our lab publication
Article Breast Cancer Stem-Like Cells Are Inhibited by Diosgenin, a ...
Article Secreted Frizzled-Related Protein 4 Inhibits Glioma Stem-Lik...
Article Cancer stem-like cells from head and neck cancers are chemos...
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