Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

Summary

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

Abstract

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level. We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination. This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain. Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells.

Protocol

Preparation before dissection

Surgical tools: sterilize all dissection scissors and forceps by autoclave

Coverslip cleaning:

1. Soak all coverslips (15mm in diameter for 24-well plate) in 1L grass beaker in 33% HCl for at least 24 h
2. Wash with running water for 10 min with occasional shake to remove all residual HCl
3. Rinse with Millipore water
4. Drain water off
5. Soak coverslips in 95-98% ethanol
6. Transfer coverslips to a clean tissue on a flat plate and air dry the coverslips

Discussion

Early studies reported formation of synapses and mature myelin in rotation-mediated free floating aggregate cultures¹. The CNS aggregate culture system described here combines serum-free growth of multipotent progenitor cells in three dimensional aggregates with the convenience of traditional 2D cultures to facilitate analyses of cell development, migration and differentiation in vitro. The system can be modified and used for neural precursor cell research and for investigation into neuron-glia intera.......