Dear all,
I culture GSCs derived from patients. Few weeks ago I started having problem with them. They dont want to grow and make spheres. They are single cells
I culture them in Neurobasal A medium with 2% B27, 10ng/ml FGF, 10ng/ml EGF and 1.5 ml of GlutaMax.
I change only the medium from Neurobasal Plus for Neurobasal A medium.
Maybe someone have got similar problem?
Dear Klaudia!
For example Glioblastoma stem cells
https://www.cell.com/cell-reports/pdf/S2211-1247(19)30242-6.pdf
Patient-Derived GSCs Following informed consent, tumor samples classified as glioblastoma, based on the World Health Organization (WHO) criteria, were obtained from patients undergoing surgical treatment at the National Institutes of Health (NIH) or from Weill Cornell Medicine/New York Presbyterian Hospital in accordance with the appropriate Institutional Review Boards. Within 1–3 hours after surgical removal, tumors were washed in PBS and enzymatically dissociated into single cells. Tumor cells were cultured in NBE medium consisting of neurobasal medium (Thermo Fisher Scientific), N2 and B27 supplements (Thermo Fisher Scientific), and human recombinant bFGF and EGF (25 ng/mL each; R&D Systems) plus Heparin sodium and L-Glutamine. Regular mycoplasma screening was performed using the MycoAlert Detection Kit (Lonza Inc.).
Ovarian adenocarcinoma cell line (OVCAR3) cells were cultured in 2D in 1640 RPMI supplemented with 10% fetal bovine serum and 1.5X antibiotics/antimycotics. Within the 3D hanging drop, OVCAR3 and Pt224 spheroids were cultured in serum-free medium, composed of 1:1 DMEM:Ham's F-12, supplemented with 1X B27 supplement without vitamin A, 1X MEM Non-Essential Amino Acids, 1X insulin-transferrin-selenium, 5 ng/mL Epidermal growth factor (EGF), and 5 ng/mL basic fibroblast growth factor (bFGF) [24], [25]. Pt412 spheroids were plated in a serum free medium that was the same as above, except for 10 ng/ml EGF and bFGF. These cells were maintained in a humidified 37°C incubator with 5% carbon dioxide.
Generating Spheroids on the Hanging Drop Array
Spheroids were formed in hanging drop plates, purchased from XCentric Mold and Engineering (Clinton Twp, MI). For initial spheroid formation, cells were counted using a hemacytometer and diluted to contain 100 cells per 20 μL, and plated in 20 μL drops, following the procedure described previously [22], [23], [24], [25]. Serum free medium (3 μL) was added to the spheroids every 2–3 days.
Article Engineered 3D Model of Cancer Stem Cell Enrichment and Chemoresistance
hi
Chapter Isolation and Culture of Glioblastoma Brain Tumor Stem Cells...
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