Abstract
Primary neuronal culture from rodents is a key tool in neurobiology. However, the preparation of primary cultures requires precise planning, starting from animal mating. Furthermore, each preparation generates a high amount of cells that eventually go wasted. The possibility to cryopreserve primary neural cells represents a resource for in vitro studies and significantly reduces the sacrifice of animals. Here we describe that Neurostore buffer supports the cryopreservation of primary neurons.
Keywords: Neurons, Cryopreservation, Long-term storage, Reproducibility, Specimen sharing
Background
Primary rodent cultures are a primary tool in any neurobiology laboratory. Primary cultures last 2-3 weeks. Therefore cells are normally prepared when necessary and employed in experiments immediately upon isolation. Cryopreservation is a routine procedure to allow long-term storage of mammalian cells. A protocol to achieve cryopreservation of neuronal cells would spare animals sacrifice and avoid the waste of cells. Furthermore, cell storage might permit the creation of cultures archive, support the reproducibility of experiments, and improve specimen sharing among different laboratories. Several studies have proposed a protocol to achieve the cryopreservation of neurons (Ichikawa et al., 2007; Paynter, 2008; Ma et al., 2010). Here we describe Neurostore, a novel buffer that promotes the long-term storage of primary mouse neurons and assures high viability upon thawing.
Materials and Reagents
Equipment
Procedure
Data analysis
Note: We refer to results and figures reported in our original publication (Pischedda et al., 2018. doi: 10.3389/fncel.2018.00081).
Notes
Neuronal mortality in Neurostore medium is estimated in 20%-30% for both cortical and hippocampal neurons if cells are stored at the concentration of 5 x 106 cells/ml Neurostore medium. It is extremely important to count cells after thawing to reduce variability between different neuronal preparations.
Recipes
Acknowledgments
Funding source: GP is a Dulbecco Telethon Scientist (Grant No. TDPG00514TA). The procedure was adapted from Pischedda et al., 2018.
Competing interests
Neurostore recipe is under patent consideration.
Ethics
All procedures involving animals were approved by Body for the Protection of Animals at the University of Trento and National Agency (autorizzazione 793/2016-PR).
References
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