Unraveling the Details of Primary Neuron Cultures: Techniques, Applications, and Yields
Introduction
Figure 1. General workflow for neuronal preparation. Figure adapted from Deshpande et al. (2016)
Primary neuronal cultures involve isolating neurons from the nervous system and growing them in a controlled laboratory environment. This technique is crucial for studying the intricate details of neuronal behavior, neurodevelopment, and neurophysiology without the complexity of the entire organism. These cultures are derived directly from the tissues of animals, typically from regions like the cortex, and are not passaged or modified, preserving their natural physiological properties.
Sourcing Neurons for Primary Culture
The process of sourcing neurons for primary culture primarily involves the use of rodents, such as mice. This process includes:
- Ethical considerations: Ensuring all animal use is approved by institutional review boards and follows ethical guidelines.
- Tissue extraction: Neurons are often sourced from specific brain regions, such as the cortex, hippocampus, or spinal cord. The brain tissue is removed from euthanized animals under sterile conditions.
- Cell dissociation: The extracted brain tissue is then enzymatically and mechanically dissociated into a single-cell suspension, ready for culturing.
Protocols for Mouse Primary Neuron Culture
Mouse primary neuron cultures are particularly popular due to the availability of genetically modified strains. Here's a basic outline of the protocol for creating primary cortical neuron cultures from mice:
- Preparation: Sterilize all instruments and prepare culture dishes coated with poly-D-lysine or a similar substrate to promote neuron adhesion.
- Dissection: Dissect the cerebral cortex from newborn mice, typically within 24 hours post-birth.
- Dissociation: Enzymatically treat the cortex to loosen the cellular matrix, then mechanically dissociate the tissue to create a suspension of individual neurons.
- Plating: Plate the neurons in pre-prepared dishes and maintain them in a controlled environment (37°C, 5% CO2).
- Maintenance: Change the medium regularly and monitor cell health and growth.
Growth Timeline in Culture
Figure 2. Timeline for cell culture procedures. Cells were obtained by exfoliation of the human olfactory epithelium and cultured in DMEM/F12 medium supplemented with 10% FBS, 2 mM glutamine, and 1% streptomycin-penicillin, at 37°C with 5% CO2. A clonal culture was obtained through the limiting dilution procedure. Red arrows indicate the precise passages from where samples were taken for experiments. hONPC: human olfactory neuronal precursor cell.
The growth of primary neurons in culture is a gradual process:
- Initial attachment: Neurons begin to attach to the substrate within the first 24 hours post-plating.
- Network formation: Over the next 7-14 days, neurons extend axons and dendrites and begin forming synaptic connections.
- Maturity: By three weeks, a well-established network of neurons exhibiting synaptic activity is usually observable.
Yield of Protein and RNA from Cultured Neurons
The yield of biological molecules from primary cultured neurons is a key concern for researchers:
- Protein yield: Depending on the number of neurons and culture density, one can typically extract about 100-200 micrograms of total protein per million cells from mature cultures.
- RNA yield: RNA extraction yields vary more significantly but generally fall in the range of 10-30 micrograms per million cells, depending on the extraction technique and the RNA stability under culture conditions.
Conclusion
Primary neuron cultures are indispensable in neuroscience, offering detailed insights into neuronal function, developmental processes, and disease mechanisms. By mastering the techniques of neuron sourcing, culture maintenance, and molecule extraction, researchers can effectively use these cultures to advance our understanding of the brain. With ongoing improvements in culture methods and ethical standards, primary neuron cultures will continue to be a cornerstone of neurological research.