Abstract
This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of cells, while offering minimal cell death that often occurs during dissociation of differentiated neurons. Cells plated under the appropriate conditions, either in Petri-dishes or in multi-well plates, will develop and establish synaptic contacts over time since the neuronal culture medium provides the nutrients and trophic factors required for differentiation. In this protocol we describe the methodology for the preparation of both cortical and hippocampal neuronal cultures.
Keywords: Primary cultures, Hippocampal neurons, Cerebrocortical neurons, Cell culture
Background
The present protocol describes the preparation of primary cultures of rat hippocampal and cerebrocortical neurons, using Neurobasal medium supplemented with NeuroCultTM SM1 (Chen et al., 2008). The composition of NeuroCultTM SM1 is based on the formulation of the B27 supplement (Brewer et al., 1993), but the former cocktail was found to improve the quality of neuronal cultures, in part by replacement of apo-transferrin with holo-transferrin (Chen et al., 2008). Furthermore, the chemical composition of NeuroCultTM SM1 was described in more detail in the original publication, allowing a better control of the experimental conditions. Neuronal cultures prepared with chemically defined culture media are characterized by the presence of a low percentage of astrocytes. The proliferation of astrocytes in cultures maintained for longer periods of time, in order to allow differentiation of neurons, is prevented by adding the chemical inhibitor of mitosis 5-Fluoro-2’-deoxyuridine.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
Work in the authors laboratory was supported by grants from the Portuguese Science and Technology Foundation (FCT), European Union–European Fund for Economic and Regional Development funding (Operational Competitiveness Program [COMPETE] grants, PEst-C/SAU/LA0001/2013-2014, POCI-01-0145-FEDER-007440, UID/NEU/04539/2013, UID/BIM/4501/2013, SFRH/BPD/84593/2012, SFRH/BPD/115546/2016 and CENTRO-01-0145-FEDER-000008: BrainHealth 2020–Early Detection, Neuromodulation and Advanced Therapies to Brain Disorders).
References
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