Neuronal cell culture involves growing neurons, the specialized cells of the nervous system, in a controlled laboratory environment outside of a living organism. This technique allows scientists to investigate the intricate biology of brain cells in isolation, providing a clearer view of their functions and interactions without the complexities of a whole organism. By maintaining these delicate cells in a dish, researchers can observe how they develop, communicate, and respond to various stimuli. This approach offers a tool for understanding the fundamental processes that govern brain activity.
Sources of Neuronal Cells
Scientists obtain neuronal cells for culture primarily through two methods. The first involves creating primary cultures, derived directly from the nervous tissue of animals, often from the brains of embryonic or neonatal rodents. These cells closely mirror neurons within a living brain, making them valuable for studying native cellular behaviors. However, primary neurons do not divide in culture, limiting their lifespan.
The second source involves immortalized cell lines and neurons derived from stem cells. Immortalized cell lines, often from neuronal tumors, divide indefinitely, providing a consistent supply of cells.
Induced Pluripotent Stem Cells (iPSCs) are a key development. These iPSCs can be generated from adult cells, such as skin or blood, and reprogrammed to an embryonic-like state. They are then guided to differentiate into specific types of neurons. This patient-specific approach allows for “disease-in-a-dish” models, enabling direct study of neurons from individuals with neurological disorders.
The Culturing Process
Establishing and maintaining a neuronal culture involves several precise steps to ensure the cells thrive. The process begins with cell isolation, where neurons are separated from their original tissue. This often involves gentle mechanical dissociation or enzymatic treatment to break down tissue structure without harming the neurons.
Following isolation, the purified neurons are plated onto a specialized cell culture vessel. These surfaces are coated with adhesion-promoting substances like poly-D-lysine or laminin, which help neurons attach and extend their processes. Without these coatings, cells struggle to adhere and form connections.
Once plated, cells require continuous feeding and incubation. They are submerged in a liquid growth medium, a formulated solution that provides nutrients, salts, vitamins, and growth factors for neuronal survival and function. Culture vessels are then placed in an incubator, which maintains a stable internal environment at 37°C, with controlled humidity and 5% CO2.
Finally, neurons need time to mature in culture, a process that can take days to several weeks. During this maturation phase, neurons extend their axons and dendrites, which are projections they use to send and receive signals. They also begin to form functional connections, known as synapses, with other neurons, allowing them to communicate and form intricate networks.
Types of Neuronal Cultures
Scientists employ various formats for neuronal cultures, each offering distinct advantages for specific research questions. The most conventional approach is two-dimensional (2D) or monolayer culture, where dissociated neurons are grown on a flat surface. This method is widely used for studying the intrinsic properties of individual neurons, such as their electrical excitability, and for examining simple cellular interactions.
A more complex model involves organotypic slice cultures, which are thin sections of living brain tissue, typically 200 to 400 micrometers thick, maintained in a laboratory setting. This technique preserves some of the brain’s original three-dimensional architecture, including local neural circuits and glial cell support structures. These slices allow researchers to study neuronal activity within a more physiologically relevant context.
Three-dimensional (3D) cultures, including organoids and neurospheres, have also been developed. In these models, neurons are either grown within a 3D scaffold or encouraged to self-assemble into small, spherical structures. These “mini-brains” offer a more accurate representation of the complex cell-to-cell interactions and spatial organization found in the actual brain, allowing for studies on neuronal migration, differentiation, and network formation.
Applications in Research and Medicine
Neuronal cell cultures are used across various fields of neuroscience research and have implications for medicine.
Disease Modeling
One application is disease modeling, particularly for neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis (ALS). By culturing neurons derived from patient iPSCs, researchers can create “disease-in-a-dish” models that exhibit hallmarks of these conditions, allowing for direct observation of cellular dysfunction and progression. This helps understand the underlying mechanisms of these complex disorders.
Drug Discovery and Toxicology Screening
The cultures also serve as a platform for drug discovery and toxicology screening. Researchers can use neuronal cultures to test thousands of potential drug compounds in a high-throughput manner, assessing their therapeutic efficacy or identifying adverse effects on neuronal health. This allows for the rapid identification of promising drug candidates that might protect neurons or restore their function, while also determining if new chemicals are neurotoxic, meaning they could be harmful to brain cells. This accelerates the development of safer and more effective treatments.
Basic Neuroscience Studies
Beyond disease-focused research, neuronal cultures are used for basic neuroscience studies. They enable scientists to dissect the processes of neuronal development, from how precursor cells differentiate into mature neurons to how they extend processes and form synaptic connections. Researchers use these cultures to investigate how neurons communicate via electrical signals and neurotransmitters, how they respond to various stimuli, and how neural networks are formed and modified. This foundational knowledge is necessary for deciphering brain function and understanding the origins of neurological disorders.