Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures

摘要

We describe the use of the neutral protease Dispase for the dissociation of neonatal rat brain tissue for the preparation of primary monolayer astrocyte cultures. The method involves 5 to 6 successive extractions with careful separation of sedimenting, undissociated tissue. This method gives an initial cell suspension of high viability (93.7±1.7 cells exclude trypan blue). In comparison trypsin (0.25) dissociated tissue gave a cell suspension that showed a lower viability of 58.2±7.6. Identical saturation densities of 1.1 to 1.2×104cells/cm2after two weeks in culture were obtained for a range of seeding densities from 1 to 4×104cells/cm2of the Dispase dissociated tissue. Staining for glial fibrillary acidic protein showed that 90–100 cells were positive for this astroglial marker. Thus, the use of Dispase for the initial dissociation of rat brain tissue seems to give primary astrocyte cultures which are very reproducible and homoge