Objective:Toexplore a safe and effective method for dissociatingneural stemcell neurospheres cultured in vitro.Methods:Neurospheres were dissociated by using of four methods

that digestion of trypsin

triturating only with pasteur pipette

grinding with stainless steel mesh and controlling volume of nerospheres with short-time digestion of trypsin.The four methods were evaluated by comparing their discrete capacity and cell survivals.Results:Trypsin digestion time prolonged led to dissociation of neurospheres

but neural stem cells(NSCs)were hardly survival.Neurospheres could not be dissociated completely by triturating only with pasteur pipette.Grinding with stainless steel mesh would damage neurospheres severely induced lowsurvival rates of NSC.In contrast

controlling volume of nerospheres with short-time digestion of trypsin could separate neurospheres better than the other methods and higher NSC survival rate(93.2%

P<0.05).Conclusion:Controlling volume of nerospheres with short-time digestion of trypsin is a safe and effective method for dissociating neurospheres of NSCs cultured in vitro better than other three methods.