S. A. Louis, C.K.H. Mak, K. Favell, S. Lloyd-Burton, E. Jervis, Vivian Lee and T.E. Thomas
Primary neuronal cultures represent a useful system in which to study neuronal biology in a simplified and controlled environment. Fully mature neuronal cultures are complex in morphology, featuring highly branched dendrites and extensive networks of synaptic connections. The peak of dendrite growth and synaptogenesis does not occur until two to three weeks in culture, with some markers of synaptic maturity requiring three weeks or longer to reach their highest expression levels. As such, the ability to establish and maintain long-term primary neuronal cultures is essential for the comprehensive study of neuronal development and function. While short-term culture of neurons can be a straightforward process, long-term (>21 days) culture of relatively pure neuronal populations is technically challenging. The development of neuron-specific serum-free media formulations was pivotal to the evolution of modern primary neuronal culture techniques, enabling neurons to be cultured in the absence of undefined serum supplements or a supporting glial cell layer. However, variability in the performance of a commonly used serum-free supplement and discrepancies in the protocols for substrate coating and cell plating have resulted in inconsistency in the quality of neuronal cultures. Variability in protocols and culture media quality becomes more problematic when cells are plated at low density or cultured long-term, because these conditions place additional stress on the cultured cells. We have developed a neuronal culture kit comprising of a neuronal basal medium (NeuroCult™ Neuronal Basal Medium) and serum-free supplement (NeuroCult™ SM1) which, when used according to optimized protocols, consistently produces high yields of functional neurons from primary mouse and rat central nervous system (CNS) tissues. NeuroCult™ SM1, based on the published B27 formulation, is optimized to more reproducibly support the survival of mature neurons in long-term culture. Here we present data on the performance of NeuroCult™ SM1 for long-term culture of embryonic rat cortical neurons.