Neuro-2a Cell Line

Neuro-2a is a mouse-driven neural cell line broadly used in neuroscience research. Mainly, these cells are employed for studying neural development, differentiation, neurotoxicity, cell signaling pathways, and axonal growth. Besides, they are used for drug development and screening purposes.

This article focuses on important aspects of Neuro-2a neuroblastoma cells that could help you before commencing work with it. It will mainly cover:

General attributes and origin of Neuro-2a cells
Neuro-2a cell line culturing information
Advantages & disadvantages of Neuro-2a cell line
Research applications of Neuro-2a cell line
Research publications featuring Neuro-2a cells
Resources for Neuro-2a Cells: Protocols, Videos, and More

1. General attributes and origin of Neuro-2a cells

This part of the article will cover the background and basic characteristics of the Neuro-2a cell line that are essential to know before working with it. Here, you will learn: What is the Neuro-2a cell line? What is the origin of the Neuro-2a cell line? What is Neuro-2a cell differentiation? What is the size of a Neuro-2a cell? What is the morphology of Neuro-2a cells?

Neuro-2a cells originated from a spontaneous tumor (neuroblastoma) of an Albino mouse (strain A). The cell line was established by R.J. Klebe and F.H. Ruddle.
The Neuro-2a neuroblastoma cells exhibit neuronal and amoeboid stem cell morphology.
Neuro-2a cell size is 12 micrometers in height [1].
The chromosome number of Neuro-2a cells may range from 59 to 193 with a modal number of 95. The karyotype of these neuroblastoma cells is unstable within a range of 94 to 98 chromosomes.

Neuronal signaling illustrated: 3D representation of neurotransmitter release in the synaptic gap by the presynaptic neuron, triggered by Ca2+ influx, leading to activated ion channels and Na+ influx in the postsynaptic neuron.

2. Neuro-2a cell line culturing information

Neuro-2a cells are widely used in neuroscience research laboratories. Before working with these cells, you should learn the imperative cell culture information mentioned below. This may help you go easy with it. You will know: What is a Neuro-2a a cell culture medium? How do you culture Neuro-2a cells? What is the doubling time of Neuro-2a cells? What is the seeding density of Neuro-2a ? What is the Neuro-2a cell culture protocol? What is the Neuro-2a cell media?

Key Points for Culturing Neuro-2a Cells

Doubling Time:	The Neuro-2a cell doubling time is approximately 70 hours.
Adherent or in Suspension:	Neuro-2a is an adherent cell line.
Seeding density:	The optimized cell seeding density for the Neuro-2a cell line is 1 x 10⁴ cells/cm². For seeding, cells are washed with magnesium and calcium-free phosphate buffer saline (1x). Later, a passaging solution (Accutase) is added, and cells are incubated for 8 to 10 minutes at an ambient temperature. After cell dissociation, fresh media is added, and cells are centrifuged. The harvested cell pellet is carefully resuspended, and cells are poured into a culture vessel for growth.
Growth Medium:	EMEM is used for culturing Neuro-2a cells. It contains 10% FBS, 2 mM L-Glutamine, 1.5 g/L NaHCO3, EBSS, 1 mM Sodium pyruvate, and NEAA for ideal cell growth. Media should be replaced 1 to 2 times per week.
Growth Conditions:	Neuro-2a cultures are maintained in a 37°C humidified incubator connected to a 5% CO2 supply.
Storage:	Cells should be stored at below -150 °C temperature in an ultra-low temperature electric freezer or the vapor phase of liquid nitrogen for the long term.
Freezing Process and Medium:	CM-1 or CM-ACF media are used to freeze Neuro-2a cells. Cells are frozen through a slow freezing process that permits only a 1 °C drop per minute. This protects cells from shock and maintains their viability.
Thawing Process:	To thaw frozen cultures, the vial is placed in a 37 °C water bath for 40 to 60 seconds until only a small ice clump remains. Then, culture media is added, and cells are subjected to centrifugation. This step helps remove freezing media components. The cell pellet is then resuspended, and the cells are transferred to a new flask filled with culture media. Afterwards, the cells are placed in an incubator at 37 °C for a minimum of 24 hours.
Biosafety Level:	Biosafety level 1 laboratory settings are obligatory to handle and maintain Neuro-2a cultures.

Microscopic image of an early passage of adherent Neuro 2a neuroblastoma cells depicting their morphology at 10x and 20x magnification.

3. Advantages & disadvantages of Neuro-2a cell line

Neuro-2a cells possess some advantages and disadvantages that distinguish them from other neuronal cell lines. Some notable pros and cons of these cells are listed here.

Advantages

The advantages of the Neuro-2a murine neuroblastoma cell line are:

Easy to Maintain:
The Neuro-2a cell line is not associated with complex or intricate cell culture procedures. They are easy to culture and maintain in research laboratories.
In Vitro Neuronal Cell Model:
Neuro-2a is a convenient neuronal cell model widely used by researchers in neuron development and differentiation studies.

Disadvantages

The disadvantages of Neuro-2a cells are:

Murine Origin:
Neuro-2a is of mouse origin, and researchers should exercise caution when extrapolating results from murine-based studies to human health and disease, as mouse and human biology can differ significantly.

4. Research applications of Neuro-2a cell line

Neuro-2a cells offer many applications in the neuroscience research field. A few promising uses of this cell line are discussed here:

Neural development: Neuro-2a is an in vitro neuronal cell model that is widely used for studying neuronal development and related processes such as differentiation, axonal growth, and synapse formation. A study conducted in 2020 utilized Neuro-2a cells to explore the role of microRNA-124 in retinoic acid-induced neuronal cell differentiation [2]. Another study investigated the Neuro 2a retinoic acid receptor gamma (RARG) and miRNA-124 expression in the neuroblastoma cell line (N2A). This study proposed that RARG is targeted by miR-124 and suppresses neurite outgrowth [3].
Neurotoxicity: Neuro-2a is also used to assess the toxic effects of drugs, chemicals, or environmental factors on neural cells. For instance, a study conducted by Zhihong Yin and co-workers investigated the neurotoxicity of bisphenol A (BPA), an environmental chemical, using Neuro-2a cells. They observed that BPA represses neuronal cell differentiation and proliferation by disrupting their cellular and synaptic integrity [4].
Drug Screening: Neuro-2a cells are also employed to evaluate the therapeutic activities of several drugs, compounds, and natural products. For example, a study used Neuro-2a cells and studied the anti-oxidative potential of Gingko biloba plant extract. Moreover, the research proposed it causes a decline in Aβ aggregation and can be a potential treatment for Alzheimer’s disease [5].

5. Research publications featuring Neuro-2a cells

Here are some interesting research papers featuring the Neuro-2a cell line.

Piperine-like alkamides from Piper nigrum induce BDNF promoter and promote neurite outgrowth in Neuro-2a cells

This study in the Journal of Natural Medicines, 2018 investigated the therapeutic effects of alkamides isolated from white pepper (P. nigrum) on Neuro-2a neuronal cells.

Nkx2. 1 downregulation is involved in brain abnormality induced by excess retinoic acid

This research in Acta Biochimica et Biophysica Sinica, 2020 proposed that the Nkx2.1 gene plays an important part in mouse brain development by regulating the Shh signaling cascade.

Exposure to bisphenol A induces neurotoxicity associated with synaptic and cytoskeletal dysfunction in neuro-2a cells

This article was published in the Toxicology and Industrial Health (2023) and proposed that an environmental chemical bisphenol A, triggers neurotoxicity in neuro-2a cells and disrupts their synaptic and cytoskeletal dysfunction.

Hexarelin Modulation of MAPK and PI3K/Akt Pathways in Neuro-2A Cells Inhibits Hydrogen Peroxide—Induced Apoptotic Toxicity

This paper in Pharmaceuticals (2021) proposed that a drug, hexarelin, modulates PI3K/AKT and MAPK cascade in neuroblastoma cells (Neuro-2a) and inhibits hydrogen peroxide-induced neuro-2a cells apoptosis.

Ethanolic fruit extract of Emblica officinalis suppresses neuroinflammation in microglia and promotes neurite outgrowth in Neuro2a cells

This research paper in Evidence-Based Complementary and Alternative Medicine (2021) proposed that Emblica officinalis fruit extract suppresses neuroinflammation in Neuro-2a cells and thus can be beneficial for neuroinflammatory diseases.

6. Resources for Neuro-2a Cells: Protocols, Videos, and More

The following are the online resources available on the Neuro-2a cells. They include Neuro-2a cell culture and transfection protocol.

Neuro-2a transfection protocol: Neuro-2a is a suitable transfection host. This link will guide you regarding the transfection protocol of the cell line. Neuro 2a transfection efficiency varies from reagent to reagent.

The following link contains the Neuro-2a cell culture protocol.

Neuro-2a cell culture protocol: This link will help you learn the neuro 2a cells subculturing protocol.
Neuro-2a cells: The website contains comprehensive knowledge about Neuro-2a cell media, seeding density, and protocols for subculturing and handling cryopreserved and proliferative cultures.

References

Lulevich, V., et al., Single-cell mechanics provides a sensitive and quantitative means for probing amyloid-beta peptide and neuronal cell interactions. Proc Natl Acad Sci U S A, 2010. 107(31): p. 13872-7.
You, Q., et al., Role of miR-124 in the regulation of retinoic acid-induced Neuro-2A cell differentiation. Neural Regen Res, 2020. 15(6): p. 1133-1139.
Su, X., et al., Retinoic acid receptor gamma is targeted by microRNA-124 and inhibits neurite outgrowth. Neuropharmacology, 2020. 163: p. 107657.
Yin, Z., et al., Bisphenol-A exposure induced neurotoxicity and associated with synapse and cytoskeleton in Neuro-2a cells. Toxicology in Vitro, 2020. 67: p. 104911.
Chen, L., et al., Gingko biloba extract (EGb) inhibits oxidative stress in neuro 2A cells overexpressing APPsw. BioMed research international, 2019. 2019.