Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Protocol

1. Compartmented culture of Rat Dorsal Root Ganglia (DRGs), Oligodendrocytes (OPCs), and hGCs (human glioma cells)

1. Preparation of compartmented culture dishes

NOTE: Perform the following steps the days before the planned harvest of the DRGs.

1. Assemble compartmented culture dishes.
2. Dilute collagen stock solution to 500 µg/mL in sterile distilled H₂O; mix thoroughly.
3. With a sterile transfer pipette, fill a 35 mm culture dish with 2 mL of collagen solution; remove the solution, leaving a thin collagen film behind, and place it into the next 35 mm dish. Repeat this process, adding more collagen solution as needed, until all dishes have been coated.
4. Once all plates are coated, place the plates in a 245 mm x 245 mm culture tray and lay three 1 mm x 1 mm gauze pads in the center of the tray.
5. To polymerize the collagen, wet gauze pads with 1 mL of concentrated ammonium hydroxide and cover the trays for 15 min.
6. Remove the gauze pads and allow the 35 mm dishes to dry in the laminar flow hood.
7. While dishes are drying, load the barrel of the syringe grease applicator with high vacuum grease. Place the compartmented chambers in a large mouth media bottle filled with distilled water. Sterilize both by autoclaving and allow to cool.
8. File off the point of an 18-G needled to make a blunt tip. Sterilize in 70% ethanol. Attach the needle to the grease syringe.
9. Sterilize the pin rake by soaking it in 70% ethanol; allow it to air-dry in the laminar flow hood.
10. Remove the lid from a dry, collagen-coated 35 mm dish. Hold the dish between the thumb and pointer finger. Hold the pin rake with the other hand. Apply a firm pressure to create even 200 µM wide scratches across the center of the dish.
11. Using a pasture pipette, place two drops of Supplemented Neuronal Basal Medium in the center of the scratches.
12. Repeat steps 1.1.10-1.1.11 until all dishes have been scratched.
13. Dry the compartmented chambers in a laminar flow hood.
14. With sterile hemostatic forceps, grasp one compartmented chamber by the center divider. Flip the hemostatic forceps so the bottom of the chamber is facing up.
15. Apply silicone grease to the compartmented chamber, starting at the top. Ensure that grease is placed neatly and overlaps at all corners.
16. Remove the lid from a 35 mm dish. Invert the dish and place the scratches over the chamber. Tap down on the bottom of the plate gently with a pair of forceps.
17. Gently flip the plate over by using the hemostatic forceps. Release the forceps.
18. Place a mound of grease at the base of the center compartment. Fill each chamber with Supplemented Neuronal Basal Medium (NB) and check for leaks. Seal leaks with silicone grease as needed.
19. Continue assembling all culture dishes and store overnight at 37°C, 5% CO₂.

Materials

Name	Company	Catalog Number	Comments
Cell Culture Dish	Corning	430165	35mm X 10mm
Hypodermic Needle, 18G	BD	511097
Ammonium Hydroxide Solution	Fisher Scientific	A669-500	Concentrated
Cell Culture Dish	Corning	430165
Campenot Chamber	Tyler Research	CAMP-10
Hemostatic Forceps	Roboz	RS-7035
Cultrex Rat Collagen I	Trevigen	3440-100-01
Pin Rake	Tyler Research	CAMP-PR
Neurobasal Medium	Thermo Fisher	21103049
silicone grease
metal file
Tray
gauze
CO2 incubator
glass pipette