Isolation and culture of ventral mesencephalic precursor cells and dopaminergic neurons from rodent brains.

The ability to isolate ventral midbrain (VM) precursor cells and neurons provides a powerful means to characterize their differentiation properties and to study their potential for restoring dopamine (DA) neurons degenerated in Parkinson's disease (PD). Preparation and maintenance of DA VM in primary culture involves a number of critical steps to yield healthy cells and appropriate data. Here, we offer a detailed description of protocols to consistently prepare VM DA cultures from rat and mouse embryonic fetal-stage midbrain. We also present methods for organotypic culture of midbrain tissue, for differentiation as aggregate cultures, and for adherent culture systems of DA differentiation and maturation, followed by a synopsis of relevant analytical read-out options. Isolation and culture of rodent VM precursor cells and DA neurons can be exploited for studies of DA lineage development, of neuroprotection, and of cell therapeutic approaches in animal models of PD. Copyright 2009 by John Wiley & Sons, Inc.