Protocol for Culturing Rat Cortical Neurons

Cortical neural cell cultures are an important model system for studying neuronal development and function, neurotoxicity screening, drug discovery, and mechanisms of neurological diseases. Proper development and survival of neurons, requires specific growth factors, signaling molecules, peptides, and vitamins.1,2 This protocol provides step-by-step instructions for dissecting and culturing cortical neurons from both embryonic (E17–E18) and postnatal (P1–P2) rat pups.

Download PDF of Illustrated Protocol

Please read the protocol in its entirety before starting.

Note: In order to yield a healthy neuron population, brain tissue from P1–P2 rat pups needs to be enzymatically digested before trituration. Extra reagents and steps are required.

Note: Aseptic techniques should be used in this protocol to ensure there is no bacterial, fungal, or mycoplasma contamination. The initial dissection and collection of the cortical tissue can be completed outside of a laminar flow cell culture hood. However, preparation of reagents and cell culture plates, and all steps following tissue harvest, should be conducted within a hood. Likewise, all reagents and materials used should be sterile.

Supplies Required

Reagents

• Antibiotic-antimycotic (100x, ThermoFisher Scientific, Catalog # 15240062), or equivalent
• Cultrex^® Mouse Laminin I (R&D Systems, Catalog # 3400-010-02)
• Cultrex^® Poly-D-Lysine (R&D Systems, Catalog # 3439-200-01)
• Deionized or distilled H₂O, sterile (dH₂O)
• L-glutamine solution (200 mM, Irvine Scientific, Catalog # 9317), or equivalent
• N21-MAX Media Supplement (R&D Systems, Catalog # AR008)
• PBS, sterile (1x): 0.137 M NaCl, 0.05 M NaH₂PO₄, pH 7.4
• Trypan blue (0.4%)

Additional Reagents for P1–P2 Rat Pups

• DNase I (Worthington Biochemical Corp., Catalog # LK003170), or equivalent
• EBSS, Ca²⁺, Mg²⁺, phenol red (ThermoFisher Scientific, Catalog # 24010043)
• Ovomucoid protease inhibitor with BSA (Worthington Biochemical Corp., Catalog # LK003182), or equivalent
• Papain (Worthington Biochemical Corp., Catalog # LK003176), or equivalent

Materials

• 15 mL conical centrifuge tube
• Cell culture plates, sterile
• E17–E18 timed pregnant rat or P1–P2 rat pups
• Ice
• Parafilm^®
• Pasteur pipette, glass, fire-polished, sterile
• Petri dishes, 60 × 15 mm
• Petri dishes, 100 × 20 mm
• Pipette tips
• Petri dishes, 100 × 20 mm
• Pipette tips

Note: Locate your desired seeding densities for your cortical neuron culture in Table 1 (pg. 30) to determine the size of the cell culture plate that should be used.

Equipment

• 37 ^°C, 5% CO₂ humidified incubator
• 37 ^°C water bath
• Autoclave
• Centrifuge
• Dissection microscope
• Dissection tools
  • Dissecting forceps, curved (e.g. Gillies)
  • Fine forceps, #5, straight
  • Forceps, #7, curved
  • Surgical scissors, small
  • Surgical scissors, large
  • Vannas-Tübingen spring scissors, straight
• Hemocytometer
• Inverted microscope
• Laminar flow cell culture hood
• Pipettes

Reagent Preparation

Note: Prepare all solutions in a laminar flow cell culture hood.

Recombinant Human BDNF (1000x)

1. Add 560 µL of Reconstitution Buffer 1 to the vial of Recombinant Human BDNF

Recombinant Human IGF-I (1000x)

1. Add 600 µL of Reconstitution Buffer 1 to the vial of Recombinant Human IGF-I

Recombinant Human IGF-I (1000x)

1. 1x N21-MAX Media Supplement,
   1x Recombinant Human BDNF,
   1x Recombinant Human IGF-I,
   1x antibiotic-antimycotic,
   0.5 mM L-glutamine in Neuronal Base Media

Procedure

Coating and Preparation of Cell Culture Plates

Note: Preparation of the culture plates should be done in a laminar flow cell culture hood.

1. Dilute the Cultrex^® Poly-D-Lysine solution with sterile dH₂O to a final concentration of 50 µg/mL.
2. Cover the wells of the culture plates with 50 mg/mL Cultrex^® Poly-D-Lysine solution (e.g., 50 µL/well for 96-well plate). Tilt the plates to ensure even coating of the well surface.
3. Incubate plates for 1 hour in a 37 ^°C, 5% CO₂ humidified incubator.
4. Aspirate the poly-D-lysine solution. Wash the wells three times with sterile dH₂O. After the third wash, aspirate the wells to completely remove all liquids.
5. Wrap plates with parafilm^® to seal. Store plates at 2–8 ^°C for up to 2 weeks.

   Note: Start the remaining steps the day before collection of the rat cortical tissue.

6. Dilute the Cultrex^® Mouse Laminin I solution with sterile PBS to a final concentration of 10 µg/mL.
7. Cover the wells of the poly-D-lysine-coated plates with 10 µg/mL Cultrex^® Mouse Laminin I (e.g., 50 µL/well for 96-well plate). Tilt the plates to ensure even coating of the well surface.
8. Incubate the plates overnight at 2–8 ^°C.
9. Aspirate the Laminin I solution from the wells prior to adding the cells. Wash the wells two times with sterile dH₂O. Aspirate the wells to remove all liquids.

Dissection of Rat Cortical Tissue

Note: Autoclave dissection tools to sterilize.

1. Warm an appropriate amount Neuronal Base Media and Complete Cortical Neuron Culture Media in a 37 ^°C water bath. Place sterile PBS on ice.

   Note: If using P1–P2 rat pups, skip to step 4.

2. Asphyxiate the pregnant rat with CO₂. Recover embryos via cesarean section using the large surgical scissors and curved dissecting forceps. Place the embryos in a 100 × 20 mm petri dish containing cold PBS. Keep the dish on ice.
3. Remove the embryos from their individual placenta sacs and wash with cold PBS.
4. Place cleaned embryos in a new 100 × 20 mm petri dish containing cold PBS. Decapitate each embryo at the head/neck junction using the small surgical scissors. Decapitate P1–P2 rat pups with the small surgical scissors
5. Place the heads in a new 100 × 20 mm petri dish containing cold PBS.
6. Stabilize the dissociated head using the #7 curved forceps and #5 fine forceps, Moving caudal to rostral, cut through the skull with the small surgical scissors.

   Note: Keep cuts shallow to avoid damaging the brain tissue.

7. Peel back the two halves of the separated skull.
8. Remove the whole brain from the head cavity using the #7 curved forceps and place in a 60 × 15 mm petri dish containing cold PBS. Keep the dish on ice. Repeat steps 4–8 for the remaining heads.
9. Put a brain in a new 60 × 15 mm petri dish containing cold PBS. Under a dissecting microscope, cut the brain with the Vannas-Tübingen spring scissors, following the median longitudinal fissure, to separate the hemispheres. Cut off and discard any brain stem tissue.
10. With the #5 fine forceps, peel off the meninges that cover each hemisphere. Open up the brain to reveal the mid-sagittal side.
11. Locate the hippocampus, which is the darker, c-shaped region, and remove it using the Vannas-Tübingen spring scissors. Place the remaining cortical tissue in a new 60 × 15 mm petri dish containing cold PBS. Keep the dish on ice. Repeat steps 9–11 for the remaining brains.
12. Using the Vannas-Tübingen spring scissors, cut the isolated cortical tissue into smaller pieces (~2 mm2).

Dissociation and Culture of Rat Cortical Neurons

Note: From this point forward, any opening of tubes/plates that contain any tissue, cells, media, or reagents should be done in a laminar flow cell culture hood.

Note: If using embryonic cortical tissue, start at step 1. If using P1–P2 tissue, start at step 3.

For embryonic cortical tissue:

1. Transfer the tissue pieces to a 15 mL conical tube. Add 5 mL of Neuronal Base Media. Gently triturate the tissue with the fire-polished Pasteur pipette until the solution is homogenous (~10–15 times).
2. Skip to step 6.

   For postnatal cortical tissue:

3. In a 15 mL conical tube, mix 20 U/mL Papain and 100 U/mL DNase I in 5 mL of EBSS. Warm the solution in a 37 ^°C, 5% CO₂ humidified incubator for 10 minutes.
4. Transfer the tissue to the 15 mL conical tube containing the warmed enzyme solution. Incubate for 20–30 minutes in a 37 ^°C, 5% CO₂ humidified incubator.
5. Gently triturate the tissue with the fire-polished Pasteur pipette until the solution is homogenous (~10–15 times).
6. Centrifuge at 200 × g for 5 minutes at room temperature. Decant the solution.
7. Resuspend the cells in…

   For embryonic cortical tissue:
   10 mL of Neuronal Base Media.
   For postnatal cortical tissue:
   5 mL of EBSS containing 1 µg/mL of Ovomucoid protease inhibitor with BSA.

8. Centrifuge at 200 × g for 4–6 minutes at room temperature. Decant the solution.
9. Wash the cells twice with 10 mL of Neuronal Base Media. Centrifuge at 200 × g for 5 minutes at room temperature. Decant the media.
10. Resuspend the cells in warmed Complete Cortical Neuron Culture Media (~10 mL). Mix 10 µL of the cell suspension with 10 µL of 0.4% Trypan blue. Count the live cells.
11. Dilute the cell suspension to the desired seeding density (see Table 1, pg. 30) with warmed Complete Cortical Neuron Culture Media. Plate the neurons on the prepared culture plates.
12. Keep cultured cortical neurons in a 37 ^°C, 5% CO₂ humidified incubator until use.

Exchanging Media in Cortical Neuron Cultures

Note: Healthy cultures can be maintained for up to 4 weeks.

1. Warm an appropriate amount of Complete Cortical Neuron Culture Media in a 37 ^°C, 5% CO₂ humidified incubator.
2. Remove half the volume of media from each well of the culture plate (e.g., remove 50 µL from each well of a 96-well plate). Gently add an equal amount of new, warmed Completed Cortical Neuron Culture Media to each well.

   Note: Do not remove all the media from the wells of the plate as this will stress the neurons.

3. Exchange the culture media every 3–4 days.

References

1. Catapano, L.A. et al. (2001) J. Neurosci. 21:8863.
2. Martin, D.L. (1992) Glia 5 :81.