Journal article
Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia
Journal of Visualized Experiments, (57)
2011
PMID: 22105098
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.
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Details
- Title
- Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia
- Creators
- Saori Shimizu - Department of Pharmacology and Physiology, Drexel University College of MedicineAnna Abt - Department of Pharmacology and Physiology, Drexel University College of MedicineOlimpia Meucci - Department of Pharmacology and Physiology, Drexel University College of Medicine
- Publication Details
- Journal of Visualized Experiments, (57)
- Publisher
- MyJove Corporation
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Pharmacology and Physiology
- Web of Science ID
- WOS:000209222200014
- Scopus ID
- 2-s2.0-83455195391
- Other Identifier
- 991014878305804721
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- Web of Science research areas
- Neurosciences