Abstract:

BACKGROUND: Neural stem cells (NSCs) cultured in vitro can form sphere due to its proliferation characteristics during suspension culture. How to isolate cell spheres into single cells is a problem facing us during subculture.
OBJECTIVE: To establish an ideal subculture method for harvesting NSCs from the rat hippocampus to obtain a large number of NSCs for investigation. 
METHODS: NSCs were isolated from neonatal 1-day rat hippocampus. After a growth of typically 5-6 days, primary neurospheres were passaged by mechanical dissociation, trypsin, TrypLE or Accutase digestion. Each method was used to dissociate neurospheres for three passages every 7 days. The ratio of cell viability and neurosphere numbers were estimated on day 1 and day 4 respectively after passage. This experiment was repeated three times.
RESULTS AND CONCLUSION: Three enzymatic dissociation each obtained single-cell suspension; while mechanical dissociation resulted in mixtures of single cells and small neurospheres. The cell viability after Accutase treatment was significantly higher than trypsin (P < 0.01) and TrypLE digestion (P < 0.05). Simultaneously, more newly formed neurospheres were generated by Accutase treatment as compared with other groups (P < 0.01). These indicated that in this experimental condition, enzymatic treatment using Accutase can be regarded as an ideal protocol for dissociating neurospheres into single cells with high cell survival which yield secondary neurospheres rapidly.