The optimized culture and identification of astrocytes from rat cortical tissue / 西安交通大学学报(医学版)

ABSTRACT

Objective To obtain highly purified astrocytes and identify the cells in each stage to support further studies.Methods The cerebral cortex of a neonatal SD rat was isolated and prepared into single cell suspension.The obtained cells were purified by differential adherence and shook at a constant temperature.By inverted phase contrast microscopy and HE staining,cell morphology was observed.The immunofluorescence staining with anti-mouse GFAP was used to identify the cells.Results The primary cortical cells developed rapidly at 3 d after culture and covered the flasks at 9-12 d.At this time,the cells showed stratification and the astrocytes lay at the lower layer.GFAP positive rate was only about (67.2 ±7.1)%.After the first passage,GFAP positive rate increased obviously (84.0±6.0)%. However, oligodendrocytes and microglias could not be removed completely,and the cells also showed stratification.Through 3 times of passages,we obtained many single species of astrocytes showing satellite shape with 2 or 3 processes,big cell body and round or oval-shaped nuclei leaned to one side.Immunofluorescence staining showed that nearly all of the cells were strong positive and the positive rate reached as high as (97.6 ± 2.4 )%.Conclusion Through differential adherence and shaking at a constant temperature,more astrocytes of high purity and in good state can be obtained.