A simple DMSO-based method for cryopreservation of primary hippocampal and cortical neurons

• Published in: Journal of neuroscience methods Vol. 333; p. 108578
• Main Authors: Ishizuka, Yuta, Bramham, Clive R.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-03-2020
• Subjects: Cell viability; Cryopreservation; DMSO; Neuronal development; Primary cultured neuron; Synapse formation; Synaptic function; Primary cultured neuron; Synapse formation; Cryopreservation; DMSO; Neuronal development; Synaptic function; Cell viability
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2019.108578

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•Cell viability of cryopreserved neurons is comparable to freshly prepared neurons.•Maturation of cryopreserved neurons is equivalent to freshly prepared neurons.•Cryopreserved neurons have functionally maturated synapses.•DMSO-containing freezing medium is suitable for cryopreservation of rat neurons.•The present method can be readily implemented as a standard laboratory practice.
Primary neuronal cultures are widely used to elucidate fundamental aspects of neuronal anatomy, physiology, cell biology, and neuronal dysfunction in animal models of disease. However, preparation of primary neuronal cultures from rodent embryos is labor-intensive, and it is often difficult to produce high-quality cultures consistently in a single laboratory, and to compare results between laboratories. To overcome these issues, cryopreservation can be used to obtain more standardized, high-quality banks of neuronal cultures.
In this study, we present a simplified cryopreservation method for rodent primary hippocampal and cortical neurons from embryonic day 18.5 fetuses, using DMSO-containing traditional cell freezing medium.
Cryopreserved neurons stored for more than 1 year in liquid nitrogen were assessed by cell imaging, as well as biochemical signaling transduction and gene expression in response to pharmacological treatments. Cryopreserved neuronal cultures were comparable to freshly prepared cultures in terms of: (1) neuronal viability, (2) neuronal morphology and maturation, (3) functional synapse formation, (4) stimulus responsiveness. These results indicate that DMSO-cryopreserved neurons are equivalent to freshly prepared neurons both developmentally and functionally.
Our method is simple and does not require special reagents or equipment.
Introduction of the cryopreserved neurons as a standard laboratory practice has the potential to increase the robustness and reproducibility of findings between laboratories and reduce the number of animals used in research.