Neural tissue-spheres: a microexplant culture method for propagation of precursor cells from the rat forebrain...

By combining new and established protocols we have developed a procedure for isolation and propagation of neural precursor cells from the forebrain subventricular zone (SVZ) of newborn rats. Small tissue blocks of the SVZ were dissected and propagated en bloc as free-floating neural tissue-spheres (NTS) in EGF and FGF2 containing medium. The spheres were cut into quarters when passaged every 10-15th day, avoiding mechanical or enzymatic dissociation in order to minimize cellular trauma and preserve intercellular contacts. For analysis of regional differences within the forebrain SVZ, NTS were derived from three rostro-caudal levels of the lateral ventricles (anterior, intermediate and posterior) and propagated separately. Explants from all three levels produced proliferating NTS, but "anterior" NTS in general grew to smaller sizes than "intermediate" and "posterior" NTS. Posterior NTS moreover maintained their neurogenic potential throughout 77 days of propagation, while the ability of anterior NTS to generate neurons severely declined from day 40. The present procedure describes isolation and long-term expansion of forebrain SVZ tissue with potential preservation of the endogenous cellular content, thus allowing experimental studies of neural precursor cells and their niche.