Neuronal cell culture involves isolating and growing neurons, the primary functional units of the nervous system, outside their natural environment in a controlled laboratory setting. This technique allows for the study of their properties and behavior. Selective media is used to achieve pure neuronal populations by promoting neuron growth while limiting the proliferation of other cell types, which is important for accurate research into neuronal development, function, and pathology.
Why Neurons Need Selective Growth Media
Culturing neurons from primary tissues presents challenges because nervous system tissue contains various cell types, including neurons, glial cells, and fibroblasts. When dissociated for culture, these non-neuronal cells often proliferate rapidly. This rapid growth can outcompete and overgrow the slower-dividing or non-proliferating neurons, making it difficult to study neuronal function specifically. Selective media addresses this by inhibiting the proliferation of non-neuronal cells, allowing for the establishment of purer neuronal cultures. This enables researchers to investigate neuronal activity and responses without confounding factors from other cell types.
How Selective Media Components Work
Selective media employ various strategies and specific components to encourage neuronal survival while suppressing the growth of other cell types.
Antimitotic Agents
Antimitotic agents, such as Cytosine Arabinoside (AraC), are commonly used in neuronal cultures at low concentrations, typically 1 to 5 µM. AraC inhibits DNA synthesis and repair in rapidly dividing cells like glia and fibroblasts, leading to their cell death. While AraC primarily affects proliferating cells, it can have neurotoxic effects on post-mitotic neurons, so its application is usually limited in duration, often to around 24 hours.
Serum-Free Conditions
Serum-free conditions are another approach to achieve selectivity. Traditional cell culture serum, such as fetal bovine serum (FBS), contains a wide range of macromolecules that promote the growth of various cell types, including fibroblasts and glial cells. Excluding or minimizing serum from the media helps prevent the overgrowth of these non-neuronal cells, leading to a more defined environment. Serum-free media also reduces the risk of contamination from infectious agents.
Specific Supplements
Specific supplements like B27 and N2 are added to serum-free basal media to provide essential nutrients, antioxidants, and growth factors tailored for neuronal health. B27 supplement promotes neuronal survival, maturation, and neural network activity, especially for long-term cultures. N2 supplement is suitable for culturing primary neurons and neural stem cells and is often used during initial culture phases to support neuronal commitment and differentiation.
Neurotrophic Factors
Neurotrophic factors, a family of biomolecules, also support the growth, survival, and differentiation of developing and mature neurons. Examples include Brain-derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3). These factors can be added to media to support neuronal health and promote specific aspects of neuronal development and function. For instance, BDNF promotes neuronal survival and can induce increased dendritic complexity.
Commonly Used Neuronal Media
Neurobasal Medium
Neurobasal Medium is a widely used basal medium for the long-term maintenance and maturation of prenatal and embryonic neuronal cells. It is often used with B27 supplement, which enhances neuronal survival and neurite outgrowth. This combination allows for long-term neuron culture without an astrocyte feeder layer, which can complicate experiments. Neurobasal Medium is a modified DMEM:F12, optimized to eliminate excitatory amino acids like glutamate and aspartate, and ferrous sulfate, which can be problematic for neuronal survival.
DMEM/F12
Another common combination is Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 Nutrient Mixture (DMEM/F12) supplemented with N2. This medium supports the growth and maintenance of various central nervous system cell types, including neural stem cells and neurons. N2 supplement, containing components like insulin, transferrin, progesterone, putrescine, and selenium, helps initiate neural differentiation and supports the initial commitment and differentiation of neurons.
Other Basal Media
Other basal media, such as Minimum Essential Medium (MEM), can also be adapted for neuronal culture, though they are less common for primary neuronal cultures than Neurobasal or DMEM/F12. These basal media are typically used as a foundation and are rarely used alone. They are combined with selective components and supplements to create an environment that supports the specific needs of neurons while controlling the growth of other cell types.