The invention provides a method for separating and culturing 
pituitary gland cells of a rat. The method comprises steps as follows: (1), an 8d male SD rat is selected and subjected to 
anesthesia execution; (2), the anesthetized rat is fixed on a board with the 
ventral side facing upwards, the 
pituitary gland of the rat is taken out under the sterile condition, put in a pre-cooled PBS (poly(butylene succinate)) liquid and washed repeatedly, bloodstain is removed, and 
pituitary gland envelope is removed; (3), the pituitary gland is 
cut into 1 mm*1 mm fragments by ophthalmic scissors in the pre-cooled PBS liquid under the sterile condition; (4), pre-heated III type 
collagenase is combined with neutral 
enzyme for mixed 
digestion; (5), the 
digestion is stopped, filtering is performed, a filtrate is collected and centrifuged, a supernatant is abandoned, a culture medium is added for resuspension, and the operation is repeated three times; (6), a 24-hole culture plate is inoculated with cells for culture; (7), the cells cultured for 7d are subjected to 
trypsin digestion and then are purified by a screen again, a supernatant is abandoned after 
centrifugation, the cells are resuspended, and the treated culture plate is inoculated with the cells; (8), the cells are subjected to 
cell immunofluorescence identification. The method for separating and culturing the pituitary gland cells of the rat is simple to operate, the obtained pituitary gland cells are high in 
survival rate and good in activity, can be used for establishing a 
cell experimental model in vitro and meet the requirement of pituitary 
gland cell experiments.