Neurocult xf basal medium

Monolayers of drNPCs were plated onto adherent Corning® CellBIND® culture dishes (Corning, Product #2394 to #3296) in either maintenance culture medium (as mentioned above) or in the NeuroCult NS-A Differentiation Kit, comprising of NeuroCult XF basal medium (catalog # 05760) and NeuroCult™ NS-A Differentiation Supplement (Human) (Component# 0574) (StemCell Technologies). Media was changed after every 2–3 days until each plate was used for PCR analysis. The differentiation commenced for 10 days, and the differentiation profile was analyzed.

Primary-reprogrammed human neural precursor cells (hereafter – directly reprogrammed neural precursor cells, drNPC) were provided by New World Laboratories Inc¹³ . Cryopreserved cells were thawed from liquid nitrogen and seeded onto laminin-coated plates in NeuroCult-XF Basal Medium (StemCell Technologies) with 1% Pen-Strep (Gibco), B-27 Supplement (50×) (Gibco), 20 ng/mL of bFGF and 20 ng/mL of EGF (complete medium). The cells were incubated at 37 °С in 5% СО₂ and 5% О₂; medium replacement was performed every 2 days. As needed, cells were passaged at a ratio of 1:3 when 80% confluence was reached.
For differentiation studies, a standard differentiation protocol was used: drNPC monolayers were switched to Neurobasal media (Gibco, USA) supplemented by Glutamax (1×, Gibco, USA), B27 (1×, Gibco, USA), CultureOne (1×, Gibco, USA), BDNF (20 ng/ml), GDNF (20 ng/ml) and Ascorbic Acid (200 µM) for 14 days.

Monolayer drNPCs were removed from the surface of culture flasks by incubation with 2 mL of Trypsin-EDTA 0.25% (Gibco) after washing with Dulbecco’s phosphate-buffered saline (D-PBS) (Gibco). After the incubation, the Trypsin-EDTA was inactivated with conditioned medium, and the suspended cells were transferred into a 50 mL tube, a cell count was performed using a TC20 automated cell counter (Bio-Rad), and the sample was centrifuged at 400 × g and 20 °С for 5 min. The supernatant was aspirated and the cell pellet re-suspended in 750 µL of the NeuroCult-XF Basal Medium (StemCell Technologies). 250 µL of the freshly prepared PRP was added to the cell suspension in NeuroCult-XF Basal Medium, resulting in a cell concentration of 4 million/mL that was then carefully re-suspended. To neutralize the sodium citrate (anti-clotting agent) in the Hemocon buffer, 2 µL of 10% CaCl₂ per 100 µL of the medium was added and the solution immediately transferred into Petri dishes with a glass cover bottom (SPL Life sciences). The gel-clot formation occurred in the Petri dishes during 20 min at 5% СО₂, 37 °С. Then, 1.5 mL of Complete medium was added to each Petri dish. The dishes were incubated at 5% СО₂, 37 °С for 2 weeks, and the medium was replaced every 2 days.