Isolation, Culture, and Characterization of Cortical and Hippocampal Neurons

The ability to culture primary neurons under serum-free conditions facilitates tighter control of neuronal studies. Some serum-free media and supplements allow for the low-density neuronal cultures, which in turn enables the study of individual neurons and synapses. This has not been possible using serum–supplemented media without a feeder layer of glial cells. In serum-supplemented media, glial cells continue to multiply, necessitating the use of cytotoxic mitotic inhibitors. Serum also contains unknown and variable levels of growth factors, hormones, vitamins, and proteins. This page details the isolation and culture of neural cells in serum-free media and supplements.

Required Materials

Isolating Rat Brain Cells

• Hibernate-E Medium (Cat. no. A12476-01)
• Gibco B-27 Plus Neuronal Culture System (A3653401)
  • Includes Neurobasal Plus Medium (A3582901) and B-27 Plus Supplement (A3582801)   
• Hibernate-E Medium, without Ca2+ (BrainBits LLC, Cat. no. HE-Ca)
• Papain (Worthington, Cat. no. LS003119)
• Trypan Blue Stain (Cat. no. 15250-061)
• Pasteur pipettes
• Hemocytometer, cell counter and trypan blue, or the Countess® Automated Cell
• Counter (Cat. no. C10227)
• Conical tubes (15-mL, 50-mL)

Culturing Embryonic Neurons

• Poly-D-lysine hydrobromide (Sigma, Cat. no. P-6407)
• 48-well plate or 8-chambered slides
• Distilled water (Cat. no. 15230-162)
• Dulbecco's Phosphate-Buffered Saline (D-PBS) (Cat. no. 14040-141)
• Gibco B-27 Plus Neuronal Culture System (A3653401)
  • Includes Neurobasal Plus Medium (A3582901) and B-27 Plus Supplement (A3582801)   

Immunocytochemistry

• Goat serum (Cat. no. 16210-064)
• Mouse anti-MAP2 antibody (Cat. no. 13-1500)
• Rabbit anti-GFAP antibody (Cat. no. 08-0063)
• Alexa Fluor 488 goat anti-mouse IgG (H+L) (Cat. no. A-11029)
• Alexa Fluor 594 goat anti-rabbit IgG (H+L) (Cat. no. A-11037)
• 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Cat. no. D1306)
• ProLong Gold antifade reagent (Cat. no. P36930)
• EM grade paraformaldehyde (Electron Microscopy Services, Cat. no. 19208)

Hibernate-E Complete Medium

Hibernate-E is a serum-free, nutrient basal medium for the short-term maintenance of cultured rat neurons and long-term storage of viable brain tissue in ambient CO₂ (0.2%) conditions. The complete medium consists of Hibernate-E medium supplemented with B-27 Plus Serum-Free Supplement.

To prepare 100 mL of Hibernate-E complete medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally.

Neurobasal Complete Medium

Neurobasal complete medium requires supplementation of Neurobasal Plus medium with B-27 Plus Supplement. Complete medium is stable for 2 weeks when stored in the dark at 2–8°C. To prepare 100 mL of Neurobasal Plus complete medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally.

For primary rat hippocampus neuron cultures, Neurobasal Plus complete medium requires additional supplementation with 25 μM L-Glutamate up to the fourth day of culture.

Coating Culture Vessels with Poly-D-Lysine

1. Prepare a 2-mg/mL poly-D-lysine stock solution in distilled water.
2. Dilute the poly-D-lysine stock solution 1:40 in D-PBS to prepare a 50 μg/mL working solution (i.e., 125 μL of poly-D-lysine stock solution into 5 mL of D-PBS).
3. Coat the surface of the culture vessel with the working solution of poly-D-lysine (150 μL/cm², i.e., 100 μL per well for a 48-well plate).
4. Incubate the culture vessel at room temperature for 1 hour.
5. Remove the poly-D-lysine solution and rinse 3 times with distilled water. Make sure to rinse the culture vessel thoroughly, because excess poly-D-lysine can be toxic to the cells.
6. Leave the coated vessels uncovered in the laminar hood until the wells have completely dried. You may use the dry plates immediately or store them at 4°C, wrapped tightly with Parafilm, for up to one week.

1. Plate ~1 × 10⁵ cells per well in poly-D-lysine coated 48-well plate or an 8-chambered slide. Bring the cell suspension volume to 500 μL per well by adding complete Neurobasal Plus medium.
   
2. Incubate the cells at 37°C in a humidified atmosphere of 5 % CO₂ in air.
   
3. Feed the cells every third day by aspirating half of the medium from each well and replacing it with fresh medium.

Preparing Paraformaldehyde Fixing Solution

20% paraformaldehyde (PFA) stock solution

1. Add PBS to 20 g of EM grade paraformaldehyde, and bring the volume up to 100 mL.
   
2. Add 0.25 mL of 10 N NaOH and heat the solution at 60°C using a magnetic stirrer until the solution is completely dissolved.
   
3. Filter the solution through a 0.22-μm filter, and cool on ice. Make sure the pH is 7.5–8.0.
   
4. Aliquot 2 mL in 15-mL tubes, freeze the tubes on dry ice, and store them at –20°C.

4% PFA for fixing

1. Add 8 mL of PBS into each 15-mL tube containing 2 mL of 20% PFA, and thaw each tube in a 37°C water bath.
   
2. Once the solution has dissolved, the tubes cool on ice.

Fixing Cells

1. Remove the culture medium and gently rinse the cells without dislodging them twice with D-PBS containing Ca²⁺ and Mg²⁺.
   
2. Fix the cells with 4% fresh Paraformaldehyde Fixing Solution (PFA) at room temperature for 15 minutes.
   
3. Rinse the cells three times with D-PBS containing Ca²⁺ and Mg²⁺.
   
4. Permeabilize the cells with 0.3% Triton®-X (diluted in D-PBS with Ca²⁺ and Mg²⁺) for 5 minutes at room temperature.
   
5. Rinse the cells three times with D-PBS containing Ca²⁺ and Mg²⁺.
   

Staining Cells

1. Incubate cells in 5% goat serum diluted in D-PBS with Ca²⁺ and Mg²⁺ for 60 minutes at room temperature.
   
2. Remove the 5% goat serum solution and incubate the cells overnight with the primary antibody (Mouse anti-MAP2 at 10 μg/mL and/or Rabbit anti-GFAP at 4 μg/mL) diluted in 5% goat serum at 4°C. Ensure that the cell surfaces are covered uniformly with the antibody solution.
   
3. Wash the cells three times for 5 minutes with D-PBS containing Ca²⁺ and Mg²⁺ (if using a slide, use a staining dish with a magnetic stirrer).
   
4. Incubate the cells with fluorescence-labeled secondary antibody (Alexa Fluor 488 goat‑anti mouse (H+L) at 10 μg/mL and/or Alexa Fluor 594 goat-anti rabbit (H+L) at 10 μg/mL) diluted in 5% goat serum solution for 60 minutes at room temperature.
   
5. Wash the cells three times with D-PBS containing Ca²⁺ and Mg²⁺. In the last wash, counter-stain the cells with DAPI solution (3 ng/mL) for 10 minutes.
   
6. Rinse the cells with D-PBS, and if desired, mount using 3 drops of ProLong Gold antifade reagent per slide and seal it with the cover slip. You may store the slides in the dark at 4°C.
   
7. Observe the cells under the microscope using filters for FITC, Cy5, and DAPI.

The thawed cortical neurons cultured in Neurobasal medium supplemented with 2% B-27 Plus Supplement show > 90% neuronal population stained with MAP2 antibody and a minimum number of astrocytes. Within 3–4 days in culture, the neurons display extensive neurite outgrowth that keeps on increasing as long as the neurons are kept healthy in culture. Note that results vary if the neurons are cultured in the presence of serum.

Figure 1. Primary Rat Cortical neurons. Immunofluorescence detection of primary neuronal cells stained with mouse anti-MAP2 antibody (Green) and presence of astrocytes as detected by rabbit anti-GFAP marker (Red). Nuclei are stained with DAPI (blue).

The procedures are designed for neuronal cells grown in Neurobasal medium supplemented with 2% B-27 Plus Supplement. Results may differ with culture systems grown in other complete media formulations, which can result in higher numbers of cells other than neurons (i.e., astrocytes).

1. Brewer GJ, Price PJ (1996) Viable cultured neurons in ambient carbon dioxide and hibernation storage for a month. Neuroreport 7:1509–1512.
2. Brewer, G. J., Torricelli, J.R., Evege EK, Price PJ (1993) Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res 35:567–576.