Protocol for Culturing Embryonic Chick Dorsal Root Ganglion Neurons

 

Dorsal root ganglion (DRGs) neurons are somatosensory neurons that reside in ganglions on the dorsal root of the spinal cord. Chick DRG culture is an indispensable model system for studying neurite outgrowth, regeneration, and degeneration, as well as the molecular mechanisms of nociception and myelination in the central and peripheral nervous systems. This protocol provides step-by-step instructions for dissecting and culturing a semi-pure DRG culture.

Download PDF of Illustrated Protocol

 

Please read the protocol in its entirety before starting.

Note: Aseptic techniques should be used in this cell culture protocol to ensure there is no bacterial, fungal, or mycoplasma contamination. The initial dissection and collection of DRGs 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

  • 70% ethanol
  • Cultrex® Mouse Laminin I (R&D Systems, Catalog # 3400-010-01) or Bovine Fibronectin Protein (R&D Systems, Catalog # 1030-FN)
  • DME medium, high glucose, no L-glutamine (IrvineScientific, Catalog # 9024), or equivalent
  • Fetal bovine serum (FBS)
  • L-glutamine-penicillin-streptomycin solution (100X), or equivalent
  • Ham’s F-12K (Kaighn’s) medium
  • HEPES buffer solution (1 M)
  • N-2 Plus Media Supplement (100X, R&D Systems, Catalog # AR009)
  • PBS, sterile (1X): 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4
  • Recombinant Human β-NGF (R&D Systems, Catalog # 256-GF)
  • Sodium pyruvate (100 mM)
  • Trypan blue (0.4%)
  • Trypsin solution (10X, Sigma-Aldrich, Catalog # T4549), or equivalent

Materials

  • 15 mL conical centrifuge tubes, sterile
  • Alcohol pads
  • Cell culture plates (96-well), sterile
  • E10-E13 embryonated Leghorn chicken eggs
  • Ice
  • Paper towel
  • Pasteur pipette, glass, fire-polished, sterile
  • Petri dishes, 60 × 15mm
  • Petri dishes, 100 mm
  • Pipette tips

Equipment

  • 37 °C, 5% CO2 humidified incubator
  • 37 °C water bath
  • Centrifuge
  • Dissecting microscope
  • Dissection tools
    • Dissecting forceps, straight (e.g. Gillies)
    • Fine forceps, #5, straight
    • Fine forceps, #5/45, angled 45°
    • Forceps, #7, curved
    • Scissors, extra narrow, straight, sharp tip
  • Hemocytometer
  • Inverted microscope
  • Laminar flow cell culture hood
  • Pipettes

 

Reagent Preparation

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

  • Dissection Media 1x L-glutamine-penicillin-streptomycin solution, 20 mM HEPES buffer solution in Ham’s F-12K medium
  • Plating Media 1x L-glutamine-penicillin-streptomycin solution, 1 mM sodium pyruvate, 10 mM HEPES buffer solution, 10% FBS in a Ham’s F-12K medium/DMEM (1:1) solution
  • Culture Media 1x L-glutamine-penicillin-streptomycin solution, 1 mM sodium pyruvate, 10 mM HEPES buffer solution, 1x N-2 Plus Media Supplement, 1 ng/mL Recombinant Human β-NGF in a Ham’s F-12K medium/DMEM (1:1) solution

 

Procedure

Coating of Cell Culture Plate

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

  1. Prepare a 15 µg/mL solution of either Mouse Laminin I or Bovine Fibronectin Protein in sterile PBS.
  2. Add 50 µL of the solution to each well of a cell culture plate. Incubate the plate overnight at 2–8 °C.
  3. Wash the wells twice with 100 µL/well of sterile PBS. Add 50 µL of culture media to each well of the cell culture plate. Incubate the plate in a 37 °C, 5% CO2 humidified incubator for 30 minutes. Plate is ready for DRG neurons to be seeded.

Dissection of Embryonic Chick DRGs

Note: Soak dissection tools in 70% ethanol for 20–30 minutes to sterilize. Place the tools on a paper towel and let air dry before use.

  1. Place sterile PBS and dissection media on ice.
  2. Wipe an embryonated egg with an alcohol pad.
  3. Holding the egg with the air sack on top, crack the eggshell. Pull the chicken embryo out using the dissecting forceps and place in a 60 × 15 mm petri dish. Decapitate the embryo using the extra narrow scissors and discard the head.
  4. With the embryo on its back, clear the spinal cord of all the visceral tissues and organs using the #7 curved forceps. Rinse the embryo cavity with sterile PBS and then dissection media.
  5. Under a dissecting microscope, dissect out the DRGs all along the spinal cord using the #5 fine forceps and place in a 60 × 15 mm petri dish containing fresh, cold dissection media. Use the #5 fine forceps and the #5/45 fine forceps to remove the extraneous tissues from the isolated DRGs.

    Note: Do not puncture the outside membrane of the DRGs.

  6. Transfer the clean DRGs to a separate 15 mL conical tube containing cold dissection media. Keep DRGs on ice until dissection is complete.
  7. Repeat steps 3–7 for 4 to 7 chicken embryos, which should provide enough DRG neurons for one 96-well cell culture plate.
  8. After all DRGs have been isolated, centrifuge the 15 mL conical tube at 193 × g for 3–5 minutes at room temperature.

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

  9. Discard the supernatant.

Dissociation and Culture of Embryonic Chick DRGs

  1. Warm an appropriate amount of plating and culture media in a 37 °C, 5% CO2 humidified incubator.
  2. Resuspend the DRGs in 4.8 mL of sterile PBS. Add 200 µL of 2.5% Trypsin with no EDTA. Mix the contents of the 15 mL conical tube by gentle agitation. Incubate the tube in a 37 °C water bath for 4–10 minutes, gently agitating the tube several times during the incubation.

    Note: The length of this trypsinization step will vary depending on the number of DRGs that were isolated. The incubation period is over once the DRGs clump together, at which point, the 15 mL conical tube can be removed from the 37 °C water bath.

  3. Add 7 mL of plating media to the 15 mL conical tube. Centrifuge at 193 × g for 3–5 minutes at room temperature. Discard the supernatant.
  4. Resuspend DRGs in 4–5 mL of plating media. Dissociate the DRG tissue into a single cell suspension by trituration with the fire-polished Pasteur pipette.
  5. Add 8–9 mL of plating media. Transfer the cell suspension to a 100 mm petri dish. Incubate the dish for 3–4 hours in a 37 °C, 5% CO2 humidified incubator.

    Note: During this incubation, non-neuronal cells will attach to the bottom of the petri dish while the DRG neurons will remain in suspension.

  6. Collect and transfer the plating media (hence, the non-attached DRG neurons) to a 15 mL conical tube. Centrifuge at 193 × g for 5–7 minutes at room temperature. Discard the supernatant.
  7. Resuspend the neurons in 2–5 mL of culture media. Mix 10 µL of the cell suspension with 10 µL of 0.4% Trypan blue. Count the live cells.
  8. Reconstitute the cells with culture media to a concentration of 15–20 × 104 cells/mL. Add 100 µL of the cell suspension to each well of a prepared cell culture plate so there are 15,000–20,000 cells/well.
  9. Keep the cultured DRG neurons in a 37 °C, 5% CO2 humidified incubator until use.

Exchanging Media in DRG Neuron Cultures

  1. Warm an appropriate amount of culture media in a 37 °C, 5% CO2 humidified incubator.
  2. Gently remove half the volume of media (i.e. 50 µL) from each well of the cell culture plate. Gently add 50 µL of new, warmed culture media to each well of the cell culture plate.

    Note: Do not remove all the media from the wells of the cell culture plate as this will cause stress to the DRG neurons.

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