Tips For Maintaining Primary Neuronal Cell Cultures Long-Term

Growing primary neurons long‐ term in vitro can sometimes prove to be a daunting task especially when your cells are highly susceptible to infection and hyperosmolarity due to medium evaporation. GS21™ Neural Supplement (GSM‐3100) has been shown to increase viability and maintain healthy primary neurons during long‐term survival. Paying close attention to culture conditions is important to ensuring that your primary neurons maintain lasting survival.  

Here we share 5 essential tips for maintaining primary neurons in culturing long-term:

Tip #1: Understand that culture conditions are crucial to maintaining neuronal health. Be consistent with your operational procedures making sure to create a very detailed protocol that you can follow with regularity.

Tip #2: Promote consistent cell density and health throughout your culture vessels by avoiding edge effects. Understand that culture conditions can be crucial to the maintaining cellular health. After plating your neurons sit them out at room temperature away from equipment vibrations for approximately 30 minutes to give cells time to adjust to their environment. Differences in cell densities can lead to variations in cellular maturity.

Tip #3: Ensure that your primary neurons are receiving the proper nutrients throughout their long-term culture. Commercially available supplements such as B27®, widely used for neuronal culture, can sometimes show variability in their ability to support neuronal survival. Based on the published NS21 supplement formulation1, GS21™, a defined neural supplement, was designed to improve the overall performance of primary neurons in culture by ensuring high quality raw materials and consistency in manufacturing. In recent studies comparing B27® Supplement with commercially available GS21™, primary neurons grown in the GS21™ Supplement showed faster maturation and increased long‐ term viability. Using a defined supplement such as GS21™ will help limit the potential harmful effects of highly variable components. Having a reliable supplemental source can drastically improve your primary neuronal cultures.

Tip #4: If primary neurons are seeded into multi‐well plates, especially 96- or 384-well plates, edge effect is very common and is often due to evaporation. A thin membrane of fluorinated ethylenepropylene (FEP Teflon® film, American Durafilm) can be placed between the lid and the plate. This membrane is permeable to O2, has no pores and is impermeable to water vapor and microbes.You will see less evaporation when incorporating FEP into your plating protocol.

Tip #5: Neurons can be very sensitive to changes in environment. Once plated, primary neurons should be left alone as much as possible, disturbing them only for media changes. Any sort of shaking or movement can prevent your neurons from growing long-term causing variability in your culture data.

[Download Tips For Maintaining Primary Neuronal Cell Cultures Long-Term as PDF]


1Chen Y, Stevens B, Chang J, Milbrandt J, Barres BA, Hell JW. NS21: Re‐defined and Modified

Supplement B27 for Neuronal Cultures. J Neurosci Methods, 171 (2) 239‐0247, 2008.

B27® is a Life Technologies registerd trademark