High fidelity cryopreservation and recovery of primary rodent cortical neurons

Cell cryopreservation improves reproducibility and enables flexibility in experimental design. Although conventional freezing methodologies have been used to preserve primary neurons, poor cell viability and reduced survival severely limited their utility. We screened several high-performance freezing media and found that CryoStor10 (CS10) provided superior cryoprotection to primary mouse embryonic cortical neurons compared to other commercially-available or traditional reagents, permitting the recovery of 68.8% of cells relative to a fresh dissection. We characterized developmental, morphometric, and functional indicators of neuron maturation and found that, without exception, neurons recovered from cryostorage in CS10 media faithfully recapitulate in vitro neurodevelopment in-step with neurons obtained by fresh dissection. Our method establishes cryopreserved neurons as a reliable, efficient, and equivalent model to fresh neuron cultures.