Preparation of dissociated mouse primary neuronal cultures from long-term cryopreserved brain tissue

Abstract Background Dissociated primary neuronal cultures are widely used as a model system to investigate the cellular and molecular properties of diverse neuronal populations and mechanisms of action potential generation and synaptic transmission. Typically, rodent primary neuronal cultures are obtained from freshly-dissociated embryonic or postnatal brain tissue, which often requires intense animal husbandry. This can strain resources when working with genetically modified mice. New method Here we describe an experimental protocol for frozen storage of mouse hippocampi, which allows fully functional dissociated primary neuronal cultures to be prepared from cryopreserved tissue. Results We show that thawed hippocampal neurons have functional properties similar to those of freshly dissociated neurons, including neuronal morphology, excitability, action potential waveform and synaptic neurotransmitter release, even after cryopreservation for several years. Comparison to the existing methods In contrast to the existing methods, the protocol described here allows for efficient long-term storage of samples, allowing researchers to perform functional experiments on neuronal cultures from brain tissue collected in other laboratories. Conclusions We anticipate that this method will facilitate collaborations among laboratories based at distant locations and will thus optimise the use of genetically modified mouse models, in line with the 3Rs (Replacement, Reduction and Refinement) recommended for scientific use of animals in research.