Background
Studying neuronal growth, development and synaptogenesis are among the hot research topics. However, it is faced with various challenges and technical limitations that include but not limited to donor's species and health, threat to life, age of embryo, glial contamination, real-time tracking, and follow-up. New method: We have successfully standardized a method for long-term primary culture of neurons collected from post-fertilized 9 day incubated chicken embryo brain overcoming the limitations mentioned above. Fertilized eggs were incubated in the laboratory and neurons from the embryonic brain were collected and low-density culture, apparently without glial contamination, was studied at least for 35 days in vitro (DIV).
Conclusions
We describe an inexpensive, simpler pure primary neuronal culture method for studying neuronal cell-biology, synaptogenesis, vesicular dynamics and it has potential to grow 3D-multilayered brain in vitro.
Methods
Every step was carried out under controlled laboratory conditions. Eggs are easily available, easy to handle, neurons from desired day of incubation could be conveniently studied for long period in apparently glia-free condition. In addition to common factors affecting primary culture, selection of culture media and cover glass coating are other key factors affecting neuronal cultures. Conclusions: We describe an inexpensive, simpler pure primary neuronal culture method for studying neuronal cell-biology, synaptogenesis, vesicular dynamics and it has potential to grow 3D-multilayered brain in vitro.
Results
Neurons were characterized by double immunostaining using stringent neuronal and glial markers. Neuronal differentiation, cytomorphology, neurite and axon formation, development and maturation, spine formation and synaptogenesis were tracked in real-time in a stage and time dependent manner. The neurons were transfected with Synaptophysin-RFP to label synaptic vesicles, which were followed in real-time under live-cell imaging. Comparison with existing methods: Every step was carried out under controlled laboratory conditions. Eggs are easily available, easy to handle, neurons from desired day of incubation could be conveniently studied for long period in apparently glia-free condition. In addition to common factors affecting primary culture, selection of culture media and cover glass coating are other key factors affecting neuronal cultures. Conclusions: We describe an inexpensive, simpler pure primary neuronal culture method for studying neuronal cell-biology, synaptogenesis, vesicular dynamics and it has potential to grow 3D-multilayered brain in vitro.