What are the components of n2 supplement?

Defined, serum-free media are now the standard in cell culture, consistently offering greater reproducibility than serum-based alternatives. The N2 supplement, a foundational medium in neurobiology, contains five core components that provide the necessary support for neural cells to thrive and differentiate in vitro.

Quick Summary

The N2 supplement is a chemically defined, serum-free cell culture medium containing insulin, transferrin, progesterone, putrescine, and selenite to support neural cells in vitro.

Key Points

• Core Ingredients: The N2 supplement is composed of five main components: insulin, transferrin, putrescine, selenite, and progesterone.

• Serum-Free Advantage: It provides a chemically defined, serum-free environment that reduces experimental variability and ensures greater reproducibility in cell culture.

• Neuronal Focus: Originally designed for neuroblastoma cell lines, N2 is now widely used to support the growth and differentiation of primary neurons from the central and peripheral nervous systems.

• Component Functions: Each ingredient has a specific role; for example, transferrin delivers essential iron, while selenite provides antioxidant protection.

• N2 vs. B27: While N2 is a foundational neural supplement, B27 is a more complex formulation containing N2's components plus additional lipids, antioxidants, and hormones.

• Proper Handling: The supplement is typically supplied as a 100x stock solution that requires careful handling, including appropriate storage conditions and sterile dilution.

The Shift to Defined Media in Neurobiology

For decades, cell culture relied on fetal bovine serum (FBS) to supply the complex cocktail of proteins, hormones, and growth factors needed for cell growth. However, FBS introduces significant experimental variability due to batch-to-batch inconsistencies in its composition. To overcome this, researchers developed chemically defined, serum-free media. The N2 supplement, based on Bottenstein's formulation, was a pioneering step in providing a more consistent and reliable environment for culturing neural cells. By defining every component, N2 allows for more controlled and reproducible experimental outcomes, which is particularly critical in sensitive neurobiological research involving neuroblastomas, primary neurons, and pluripotent stem cell differentiation.

The Core Components of N2 Supplement

An understanding of what are the components of n2 supplement is essential for researchers. The formulation consists of five key ingredients, each playing a crucial role in supporting the growth and function of neural cells. This precise mix creates a selective environment that favors the survival and differentiation of neuronal cells while suppressing non-neuronal cells like fibroblasts.

• Insulin: A polypeptide hormone that plays a critical role in cellular metabolism, growth, and survival. In cell culture, insulin enhances glucose uptake and promotes cell proliferation. Different versions of the supplement may use bovine insulin or recombinant human insulin, with comparable performance.
• Transferrin: This iron-binding glycoprotein is essential for the transport of iron into cells. Iron is a cofactor for many metabolic enzymes and is vital for cell respiration and growth. Transferrin, often used in an iron-saturated (holo) form, ensures that cells receive the necessary iron without excess free iron, which can be toxic.
• Putrescine: A polyamine involved in cell proliferation and differentiation. Polyamines are crucial for DNA synthesis, cell growth, and are known to protect cells from oxidative damage. Putrescine helps maintain the health and viability of the neural cultures.
• Sodium Selenite: A source of the trace element selenium. Selenium is a key component of several antioxidant enzymes, including glutathione peroxidase. Its presence in the N2 supplement helps protect cells from oxidative stress and is critical for maintaining cell integrity in vitro.
• Progesterone: A steroid hormone known to promote the maturation and stability of neural cells in culture. It plays a role in cellular signaling pathways that support neuronal differentiation and survival, contributing to the overall stability of the culture environment.

N2 vs. B27 Supplement: A Comparative Look

N2 and B27 are both popular serum-free supplements used in neurobiology, but they were designed for different purposes and contain distinct components. Understanding their differences helps researchers choose the most appropriate medium for their specific applications.

Feature	N2 Supplement	B27 Supplement
Core Components	Insulin, Transferrin, Putrescine, Selenite, Progesterone.	Contains all N2 components plus additional factors including thyroid hormone T3, fatty acids, and antioxidants like vitamin E and glutathione.
Key Differentiators	Simpler, more basic formulation, selective for neuronal cells.	More complex, with additional components designed for broader applications and long-term cultures.
Primary Application	Originally designed for neuroblastoma cell lines and initial differentiation of neural progenitor cells.	Developed for long-term culture and maintenance of primary embryonic neurons.
Serum-Free Status	Chemically defined and serum-free.	Chemically defined and serum-free.
Versatility	Excellent for studies focused on early commitment and differentiation.	Supports a wider range of neuronal cell types and conditions, often used for maturation and maintenance.

Advantages of Using a Defined N2 Medium

The move from serum-based media to defined supplements like N2 has numerous advantages for research, particularly in fields requiring high reproducibility and consistency.

• Reduced Experimental Variability: By eliminating the undefined components found in serum, N2 provides a consistent and reproducible cell culture environment. This is critical for generating reliable data in experimental studies.
• Improved Control: A chemically defined medium allows researchers to control and precisely modify the cellular environment. This is invaluable for investigating the effects of specific factors on neural cell behavior, growth, and differentiation.
• Enhanced Cell Purity: N2's selective formulation favors the growth and survival of neuronal cells, making it easier to maintain pure neural cell cultures without overgrowth from non-neuronal cell types.
• Safety and Reduced Contamination: Serum-free media like N2 minimize the risk of contamination from infectious agents present in animal-derived serum.
• Support for Specific Applications: N2 is optimized for specific tasks, such as differentiating pluripotent stem cells into neural lineages or culturing specific neuroblastoma cell lines, providing targeted nutritional support for these applications.

How to Use and Handle N2 Supplement

N2 supplement is typically supplied as a 100x concentrated stock solution that is sterile-filtered and ready for use. It is important to handle it correctly to ensure its efficacy and to prevent contamination.

Preparation and Storage Guidelines

1. Thawing: Thaw the frozen N2 stock naturally in a refrigerator at 2°C to 8°C. Avoid repeated freeze-thaw cycles, which can degrade the components. It can be aliquoted into smaller volumes to minimize this.
2. Dilution: Dilute the 100x concentrate into a basal medium, such as DMEM/F12 or Neurobasal medium, to the final working concentration (1x).
3. Mixing: Gently mix the components. Avoid excessive agitation. Depending on the application, additional growth factors like bFGF or EGF may be added.
4. Storage: Store the concentrate at -20°C and protect it from light. The diluted working medium should be stored at 2°C to 8°C in the dark and used within a few weeks.

Applications in Research

Beyond its initial use for neuroblastomas, the N2 supplement's defined composition has expanded its utility. It is now widely used in laboratories for:

• Primary Neuronal Culture: Supporting the survival and expression of neurons from the central and peripheral nervous systems.
• Pluripotent Stem Cell Differentiation: Guiding pluripotent stem cells (PSCs) towards a neural fate, a crucial step in creating cell models for disease research.
• Neural Progenitor Cell Expansion: Promoting the reproducible expansion and differentiation of neural progenitor cells.
• Drug Discovery and Screening: Providing a reliable and standardized environment for testing compounds on neuronal cells, reducing the variability that could mask or alter results.

Conclusion

In summary, the N2 supplement is a powerful and essential tool for modern neurobiological research. Its defined composition, featuring insulin, transferrin, putrescine, selenite, and progesterone, offers a significant improvement in reproducibility and control over traditional serum-based media. By providing a targeted and consistent environment, N2 supports the growth, differentiation, and survival of various neural cell types, enabling more reliable experimental outcomes and driving forward critical research in neuroscience and regenerative medicine. The careful preparation and correct application of these specific components are key to its success in the lab.

Additional Resources

For further information on N2 supplement and its applications, consult manufacturer's guidelines and detailed protocols, such as those available on the R&D Systems website.

Frequently Asked Questions

What is the primary function of the N2 supplement in cell culture?

The N2 supplement is used to support the growth, survival, and differentiation of neural cell types, including neuroblastomas, primary neurons, and pluripotent stem cells.

Why is a serum-free medium like N2 preferred over a serum-based medium?

N2 provides a chemically defined environment, eliminating the variability found in animal-derived serum. This leads to more consistent, reproducible experimental results and reduces the risk of contamination.

What is the difference between N2 and B27 supplements?

The key difference is composition. B27 includes all N2 components but adds additional factors like fatty acids, antioxidants (e.g., vitamin E), and thyroid hormone (T3), making it better for long-term neuronal cultures.

How should I store N2 supplement to maintain its efficacy?

N2 supplement is best stored frozen at -20°C in the dark. To prevent degradation from freeze-thaw cycles, it can be aliquoted into smaller volumes.

Is there a difference between N2 and N2 Plus media supplements?

Yes, some variations exist between manufacturers. For example, some 'N2 Plus' versions use bovine insulin, while 'N2 Max' versions might use recombinant human insulin, though their performance is often comparable.

What basal medium is typically used with N2 supplement?

N2 supplement is most commonly used with basal media such as DMEM/F12 or Neurobasal media, depending on the specific cell type and application.

Does N2 supplement contain any animal products?

While the formulation is serum-free, older versions or some alternatives may use components derived from animal sources, such as bovine insulin. Modern versions often use recombinant human alternatives to be fully animal origin-free.

Can I use N2 for applications other than neural cells?

While optimized for neuronal cells, N2 has also been successfully used to differentiate pluripotent stem cells into other lineages, such as pancreatic-like cells, in combination with other factors.