Neurons, the fundamental cells of the nervous system, are delicate and challenging to culture in vitro. Early neuronal culture relied on serum-containing media, which provided nutrients but introduced significant variability due to their undefined composition. This made consistent and reproducible experimental results difficult. The scientific community later shifted towards chemically defined, serum-free supplements, allowing for greater control and more reliable outcomes. The B-27 supplement emerged as a widely adopted standard for supporting neuronal growth and survival. The B-27 Plus Neuronal Culture System represents an advanced development of this standard, designed to address the increasing demands of complex and extended neural studies. It aims to provide an optimized environment for sustained neuronal health and function.
Composition of the B-27 Plus Supplement
The original B-27 supplement provided a foundational blend of components to support neuronal health in culture. This included vitamins like biotin and vitamin E for cell metabolism and antioxidant defense. Lipids like cholesterol were incorporated as building blocks for cell membranes and neuronal signaling. Antioxidants such as glutathione were present to combat cellular stress, alongside biomolecules like insulin and transferrin for nutrient uptake and iron transport. The “Plus” in the B-27 Plus supplement signifies enhancements to this classic formulation. While proprietary, the updated version includes additional, non-disclosed antioxidants and specialized compounds. These additions provide further protection to neurons in the culture environment. The newly added components target pathways associated with oxidative stress, a common challenge for neurons in vitro, and promote synaptic development. These modifications create a more robust and supportive environment for long-term neuronal maintenance. The overall formulation reflects an upgraded manufacturing process and more stringent quality control of raw materials, contributing to its improved performance.
Functional Advantages for Neuronal Viability and Maturation
The B-27 Plus Neuronal Culture System offers significant improvements in maintaining neuronal health over extended periods. Its enhanced antioxidant profile helps shield neurons from damaging reactive oxygen species, leading to a higher survival rate in cultures maintained for several weeks. This extended viability allows longer-term studies on neuronal development and disease progression. The supplement also promotes accelerated and more extensive neuronal maturation. This is evident through increased neurite outgrowth, where neurons develop longer and more complex projections, and enhanced arborization, the intricate branching of dendrites that allows for more connections. Neurons cultured in this system also show rapid development of functional, electrophysiologically active synapses, meaning they form connections and transmit electrical signals effectively. The system additionally provides increased protection against excitotoxicity, where excessive neuronal stimulation leads to cell damage, and phototoxicity from light exposure during imaging.
Applications for Specific Neuron Types
The B-27 Plus Neuronal Culture System finds widespread utility across various neuroscience applications, particularly for primary central nervous system (CNS) neurons. It effectively supports the growth and maintenance of common models such as hippocampal, cortical, and cerebellar neurons derived from rodents. This broad applicability makes it a versatile choice for many research studies. The system is also well-suited for culturing human and rodent stem-cell-derived neurons, including those differentiated from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). These stem cell-derived neurons require robust and consistent conditions for their differentiation into mature neuronal phenotypes and long-term maintenance in vitro. Its ability to support these sensitive cell types stems from its optimized formulation, which addresses the high metabolic demands and inherent sensitivity of developing neurons. This enhanced support contributes to the successful maturation and functionality of stem cell-derived neurons, enabling more biologically relevant models for studying human neurological conditions.
Implementation in Culture Systems
The B-27 Plus supplement is supplied as a 50X concentrate, requiring significant dilution before use. It is designed to be added to a compatible basal medium, forming a complete neuronal culture medium. The most commonly used basal media for this purpose are Neurobasal Medium and Neurobasal Plus Medium. Proper handling of the supplement is important to maintain its efficacy. The concentrated B-27 Plus supplement should be stored frozen at -20°C to preserve its components. When preparing the culture medium, the frozen supplement should be thawed gently, either at room temperature for about an hour or overnight at 2°C to 8°C; thawing at 37°C is not recommended. To avoid repeated freeze-thaw cycles, which can degrade sensitive components, aliquot the thawed supplement into smaller volumes for future use. The final working concentration of B-27 Plus in the complete culture medium is 1X. Researchers often add other factors, such as L-glutamine or GlutaMAX supplement, separately to the basal medium to support optimal neuronal growth and survival.