Cryopreservation of Primary Mouse Neurons: The Benefit of Neurostore Cryoprotective Medium

Abstract

Primary neuronal culture from rodents is a well-established model to investigate cellular neurobiology in vitro. However, for this purpose cell cultures need to be generated expressly, requiring extensive animal handling. Furthermore, often the preparation of fresh culture generates an excess of cells that are ultimately wasted. Therefore the ability to successfully cryopreserve primary neural cells would represent an important resource for neuroscience research and would allow to significantly reduce the sacrifice of animals. We describe here a novel freezing medium that allows long-term cryopreservation of primary mouse neurons prepared from E15.5 embryos. Combining imaging, biochemical and electrophysiological analyses, we found that cryopreserved cultures are viable and mature regarding morphology and functionality. These findings suggest that cryopreserved neurons are a valuable alternative to acutely dissociated neural cultures.

High Fidelity Cryopreservation and Recovery of Primary Rodent Cortical Neurons

“…Using whole-cell patch clamp electrophysiology, we observed similar examples of spontaneous and evoked APs elicited by fresh and cryostored neurons, in agreement with previous reports that the number, burst duration, and amplitude of APs are not significantly affected following cryostorage in several media ( Otto et al, 2003 ; Quasthoff et al, 2014 ; Pischedda et al, 2018 ). Additionally, careful analysis of specific features of evoked AP waveforms did not detect significant differences after recovery from cryostorage.…”

“…In contrast, previous methods found, at best, a 40% reduction in long-term survival compared to freshly dissected controls ( Mattson and Kater, 1988 ; Higgins et al, 2011 ). A recent publication using another pre-commercial, proprietary freezing media formulation also demonstrated improvements in cell viability and culture health compared to traditional freezing media ( Pischedda et al, 2018 ). This underscores the importance of reagent choice when applying cryopreservation methodologies to primary neuron culture.…”

Ankyrin-G induces nucleoporin Nup358 to associate with the axon initial segment of neurons

“…Cortical and hippocampal cultures were prepared from E15 and E17 mouse embryos, respectively, using a previously described method (Qian et al, 1998;Pischedda et al, 2018) with some modifications. Briefly, cortices or hippocampi were dissected from C57BL/6 mouse brains and treated with papain solution [ papain (20 U, Worthington), EDTA (5 mM), and cysteine (30 mM) in 1× Earle's balanced salt solution (EBSS, Gibco)] for about 20 min in a 37°C water bath, with occasional swirling of the mixture.…”

The LRRK2 N-terminal domain influences vesicle trafficking: impact of the E193K variant

“…N2A (Neuro2a, ATCC CCL-131) cells were grown in DMEM supplemented with 10% FBS, 1% penicillin/streptomicin and 1% glutamine in a humidified atmosphere of 5% CO 2 at 37 °C. Cortical cultures were obtained from embryonic day 15.5-16.5 mouse as described 37,38 . N2A cells and DIV4 neurons were transfected with the different constructs using Lipofectamine 2000 (Invitrogen).…”