Because NPCs represent a convenient starting point for in vitro neural differentiation, a conventional DMSO-based, controlled-rate cooling program was applied to the adherent cells described previously with subsequent storage in liquid nitrogen. On thawing, 75.4% ± 8.7% (four replicates) of the NPCs retained viability as evidenced by trypan blue staining. In two further replicates, a total of 41 free-floating spherical structures were equally divided and stored at low temperature using vitrification or slow-rate controlled freezing. After thawing and culture in N2 media, 20 of 25 vitrified spherical structures (80%) survived and attached. Twelve of 16 conventionally frozen spherical structures (75%) also survived and attached. buy zyban
Thus, these methods of low-temperature storage yield comparable viabilities after thawing. Additionally, on FGF-2 removal, these NPCs stored at low temperature showed the same ability as nonfrozen NPCs to differentiate into neuronal lineages as described below.
Neuronal and Glial Cell Differentiation
Differentiation of NPCs into neuronal and glial cell lineages was initiated by removal of FGF-2 from the NPC culture medium and plating cells onto polyornithine and laminin substrates.