L glutamine

The human ovarian cancer cell line SKOV3 was purchased from the Korean Cell Line Bank. The cells were cultured and maintained in the Roswell Park Memorial Institute-1640 (RPMI-1640) medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 1% penicillin/streptomycin (HyClone), and 25 mM HEPES (Gibco).
Human glioblastoma (GBM) cell lines A1207, LN18, LN229, T98G, and U87MG and the human cervical cancer cell line HeLa were purchased from the American Type Culture Collection (ATCC). These cell lines were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (4500 mg/L) (HyClone), supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, and 50 µg/mL gentamicin (Tocris, Bristol, UK). All cells were incubated and maintained at 37 °C in a humidified 5% CO₂ chamber.

Prior to differentiation, cells were dissociated with Accutase™ (StemCell Technologies, Vancouver, BC, Canada) and passaged to six-well plates. Until 100% confluence of iPSCs, NutriStem™ (Biological Industries) was refreshed. On day 0, the medium was changed to 3 mL of RPMI (Biological Industries), supplemented with 0.5% L-glutamine (Biological Industries), B27-Insulin (Invitrogen, Carlsbad, CA, USA) and 4.5 µM CHIR-99021 (Tocris, Bristol, UK). On day 2, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine, B27-Insulin, and 5 µM IWP-2 (Tocris). On day 4, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine and B27-Insulin. This medium was refreshed on day 6. On day 8, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine and B27, and this medium was refreshed on day 10. From day 12, the medium was changed to M-199 (Biological Industries), supplemented with 0.1% penicillin/streptomycin, 5% fetal bovine serum (FBS, Biological Industries), 0.6 mM CuSO₄·5 H₂O, 0.5 mM ZnSO₄·7 H₂O, and 1.5 mM Vitamin B12 (Sigma-Aldrich, Darmstadt, Germany). This medium was refreshed every other day.

Prior to differentiation,
cells were passed to 6-well plates. NutriStem was refreshed daily
until iPSCs reached 100% confluence. At that point (Day 0), the medium
was changed to 3 mL of RPMI (Biological Industries), supplemented
with 0.5% l-glutamine (Biological Industries), B27-Insulin
(Invitrogen, Carlsbad, California), and 4.5 μM CHIR-99021 (Tocris,
Bristol, UK). On Day 2, the medium was changed to 3 mL of RPMI supplemented
with 0.5% l-glutamine, B27-Insulin, and 5 μM IWP-2
(Tocris). On Day 4, the medium was changed to 3 mL of RPMI supplemented
with 0.5% l-glutamine and B27-Insulin, and this medium was
refreshed on Day 6. On Days 8 and 10, the medium was changed to 3
mL of RPMI supplemented with 0.5% l-glutamine and B27. From
Day 12, the medium was changed to M-199 (Biological Industries), supplemented
with 0.1% penicillin/streptomycin, 5% fetal bovine serum (FBS, Biological
Industries), 0.6 mM CuSO₄·5H₂O, 0.5 mM
ZnSO₄·7H₂O, and 1.5 mM Vitamin B12 (Sigma-Aldrich).
This medium was refreshed every other day.^{74 (link)}