What Does N2 Supplement Do for Neural Cell Culture?

December 22, 2025 •

2 min read

In neuroscience, over 90% of cells in a healthy brain are neurons or glial cells, underscoring the need for specialized media to study them effectively. This is where the N2 supplement becomes critical, as it is a chemically defined, serum-free additive designed specifically to support the growth and survival of neuronal cell types in vitro.

Quick Summary

N2 supplement is a serum-free, chemically defined media additive that promotes the growth, differentiation, and survival of various neural cells, including neurons and neuroblastomas. It eliminates the experimental variability associated with serum-containing media, providing a more consistent and controlled environment for neuronal culture studies.

Key Points

Serum-Free Media: N2 supplement is a chemically defined, serum-free additive used for culturing specific neural cells.
Neural Differentiation: It is primarily used to support the growth and initial differentiation of neurons and neuroblastomas.
Key Components: The formulation includes recombinant human insulin, human transferrin, progesterone, putrescine, and sodium selenite.
Reduces Variability: By eliminating undefined serum, N2 minimizes experimental variability and ensures more consistent, reproducible results.
Not for Long-Term Culture: While ideal for initial differentiation, B27 supplement is often preferred for long-term neuronal survival.
Application in Stem Cells: N2 is critical for directing the differentiation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into neural types.
Protects Against Stress: Sodium selenite in the formulation acts as an antioxidant, protecting cultured cells from oxidative damage.

Understanding the Core Function of N2 Supplement

N2 supplement is a specialized, serum-free additive used primarily in biomedical and neuroscientific research to create a chemically defined environment for cell culture. It provides a precise blend of essential factors for neural cell growth. Standard N2 formulations contain recombinant human insulin, human transferrin, progesterone, putrescine, and sodium selenite.

The Role of N2 Components

These components each play a vital role:

Recombinant Human Insulin: Promotes metabolic health and survival.
Human Transferrin: Transports iron, essential for cellular processes.
Progesterone: Supports maturation and stability of neural cells.
Putrescine: Involved in DNA synthesis, growth, and differentiation.
Sodium Selenite: Provides selenium for antioxidant defense.

Supporting Neural Cell Types

N2 supplement is particularly effective for culturing specific neural cell types. It was originally developed for neuroblastoma cell lines and is now used for primary neuron cultures from both the PNS and CNS. It is selective, inhibiting non-neuronal cell growth, and supports initial differentiation of post-mitotic neurons.

Comparison: N2 vs. B27 Supplement

N2 and B27 supplements are both used in neural cell culture but have different primary applications.


Feature	N2 Supplement	B27 Supplement
Primary Use	Initial differentiation and short-term survival.	Long-term survival of mature neurons.
Composition	Defined components like insulin, transferrin, progesterone, putrescine, selenite.	Defined components plus thyroid hormone T3, fatty acids, antioxidants.
Cell Selectivity	Favors neuronal growth, inhibits non-neuronal cells.	Supports neuronal growth, broader range of mature neural cells.
Culture Phase	Initial differentiation phase.	Later stages for mature neuron growth.
Base Medium Compatibility	Often with DMEM/F12 or Neurobasal Medium.	Often with Neurobasal Medium for long-term culture.

The Mechanism of Action

N2 components work together to create a controlled environment mimicking conditions for neuronal development. Transferrin provides iron, insulin aids glucose metabolism, and putrescine and progesterone support growth and differentiation towards a neuronal fate. The absence of serum eliminates variability and minimizes non-neuronal growth, leading to more reproducible cultures.

Application in Stem Cell Research

N2 is crucial for differentiating ES and iPS cells into neural types, enabling disease modeling and therapeutic development in a controlled environment. Its defined nature ensures predictable differentiation.

Benefits in Research

Consistency: Defined composition reduces variability.
Specificity: Promotes neuronal cell growth.
Control: Allows for precise manipulation and observation.
Differentiation: Supports initial commitment to neuronal phenotypes.

Limitations and Considerations

N2 is optimized for specific cell types and early differentiation stages. For long-term or mature neuronal cultures, supplements like B27 may be better. Combining N2 with other supplements, like G5, can enhance neural differentiation.

Conclusion

N2 supplement is a vital tool in neuroscience research, enabling controlled in vitro culture of neural cells. Its serum-free, defined composition reduces variability and supports initial differentiation and survival of neurons and neuroblastomas. The blend of insulin, transferrin, progesterone, putrescine, and selenite ensures metabolic health and controlled growth. Understanding N2's specific role and its differences from supplements like B27 is key for accurate and reproducible results in studying the nervous system.

Frequently Asked Questions

The primary purpose of N2 supplement is to provide a chemically defined, serum-free environment for the culture of neuronal cell types, including neuroblastomas and post-mitotic neurons from the peripheral and central nervous systems.

Key ingredients typically include recombinant human insulin, human transferrin, progesterone, putrescine, and sodium selenite, which collectively support neural cell growth and survival.

N2 is used in stem cell research to facilitate the differentiation of embryonic stem cells and induced pluripotent stem cells into specific neural and pancreatic cell types, providing a controlled environment for studying cell lineage commitment.

N2 is used for initial differentiation and shorter-term survival, while B27 is better suited for long-term maintenance of mature neurons. B27 also contains additional components like fatty acids and vitamin E.

No, N2 supplement is selective for neuronal cell lines and is not recommended for culturing non-neuronal cell types, as it may not support their growth.

A serum-free supplement like N2 is advantageous because it provides a consistent, defined culture environment, which reduces the experimental variability associated with using undefined, serum-based media.

N2 supplement is typically stored as a 100x concentrate at -5 to -20°C and protected from light. It should be thawed and diluted with an appropriate basal medium before use.