Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord

1. Preparation of Solutions in a Culture Hood under Aseptic Conditions

1. Prepare culture media: complete Dulbecco's modification of Eagle's media (cDMEM) containing DMEM (with 4.5 g/L glucose), 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, 250 ng/mL Amphotericin B, and 50 µM 2-mercaptoethanol (2-ME; see 1.1.1). Mix and filter sterilize all other components and then add FBS. Store the culture media at 4 ^°C.
   1. Prepare 50 mM 2-ME/phosphate-buffered saline (PBS) stock solution: add 100 µL of concentrated 2-ME (14.3 M) into 28.6 mL of 1x PBS, and filter sterilize (the solution can be stored at 4 ^°C for several months). Use 1 mL of 2-ME stock solution for every 1,000 mL of culture media.
2. Preparation of the density gradient media
   1. Prepare a stock isotonic density gradient media solution (e.g., 100% Percoll) from 1 part 10x regular sterile PBS and 9 parts sterile stock density gradient media (e.g., Percoll).
   2. Dilute the 100% density gradient media (made in 1.2.1) with regular 1x sterile PBS to make a 20% density gradient media (e.g., 10 mL of 100% density gradient media with 40 mL of 1x PBS) and store it at 4 ^°C. Bring the 20% density gradient media to room temperature (RT) before preparing the cell culture.
3. Preparation of solutions from the papain dissociation system
   ​NOTE: The papain dissociation system can be identified in the Materials Table. Other similar dissociation kits (including those assembled in-house) can also be used. All components must be sterile.
   1. Add 32 mL of Earle's Balanced Salt Solution (EBSS) to the ovomucoid inhibitor mixture (powder).
   2. Add 5 mL of EBSS to one papain vial (powder). Place the papain vial in a 37 ^°C water bath for 10 min or until the papain is dissolved.
   3. Add 500 µL of EBSS to one DNase vial (powder). Mix gently.
   4. Add 250 µL of the DNase solution (above) to the papain vial.
   5. Calculate the total amount of the above papain/DNase mixture needed (approximately 800 µL of papain/DNase mixture per mouse spinal cord) and transfer the needed mixture into a 50 mL tube for tissue digestion. Store the leftover in the original vial at 4 °C for at least two weeks.
   6. Keep all components from the kits on ice until needed.
4. Prepare spinal cord collection tubes: one sterile 15 mL tube with 5 mL of Hank's balanced Salt Solution (HBSS; from the papain dissociation system, for collecting spinal cords) and one sterile 15 mL tube with 1x PBS (for filling up the 5 mL syringe). In addition, prepare one sterile petri dish (35 mm or 60 mm) with 5 mL of HBSS and keep it in the culture hood.

2. Preparation of the Single Cell Suspension

Note: Perform steps 3 and 4 in a culture hood to keep everything sterile.

1. Transfer all spinal cords into the HBSS-containing petri dish. Cut each of the spinal cords into many fine, small pieces with sterile scissors and forceps. Transfer the spinal cord tissue to the 50-mL conical tube containing the prepared papain/DNase enzyme mixture (step 1.3.5) using sterile forceps or a 10 mL pipette (avoid adding HBSS to the enzyme mixture, as this will further dilute the prepared enzyme solution and may result in decreased performance of the enzyme).
2. Vortex the tube gently to mix. Incubate the tube at 37 ^oC for 1 h in an incubator/shaker with orbital shaking at 150 rpm.
3. Vortex the tube again and vigorously triturate the enzyme solution with the tissue using a 5 mL pipette to promote further dissociation.
4. Transfer the cell suspension into a 15-mL tube and centrifuge at 300 x g for 5 min at RT.
5. During centrifugation, mix 2.7 mL EBSS with 300 µL of reconstituted albumin-ovomucoid inhibitor solution (1.3.1) in a sterile tube. Add 150 µL of the DNase solution (step 1.3.3).
6. Following centrifugation, remove the supernatant and resuspend the cell pellet with the solution prepared above (step 1.5). Vortex well to break the cell pellet.
7. Add 3 mL of reconstituted albumin-ovomucoid inhibitor solution (step 1.4.1) to the cell suspension. Centrifuge cells at 70 x g for 6 min at RT. Remove the supernatant (which contains membrane fragments).

3. Further Removal of Myelin from the Single Cell Suspension

1. Add 8 mL of 20% density gradient media (prepared in step 1.2.2) into the tube containing the cell pellet, vortex gently to disrupt the pellet, and centrifuge the cells at 800 x g for 30 min at RT without braking. Carefully remove the top layer of debris (mostly myelin) and the supernatant but keep the pellet.
2. To remove remnants of the density gradient, wash the cells by resuspending the cell pellet with 8 mL of a diluted cDMEM (1 part cDMEM and 2 parts HBSS). Centrifuge the cells at 400 x g for 10 min at 4 ^oC. Remove the supernatant and wash the cells again with the diluted cDMEM (above) in the same manner. Keep the cells on ice until seeding them.
3. Remove the supernatant and resuspend the cell pellet in culture media (cDMEM supplied with 2-ME (prepared in step 1.1)). For one 12-well plate, use 3 mL x 4 (number of mice used) + 2 mL = 14 mL media. This will ensure that there is sufficient cell suspension for the entire plate (12 wells) and will provide for extra wells that can be used to determine the average cell number per well and the microglial content of the culture. If other types of culture vessels will be used, calculate the total volume of needed culture media proportionally.
4. Add 1 mL of the cell suspension into each well of a 12-well plate.
5. Incubate the cells at 35.9 ^oC with 5% CO₂.
6. Change the media (remove old media via aspiration) on D 1 and then every 3 – 4 d thereafter (typically change the media on D: 1, 4, 8, and 11, and then use the cells on day 12).
   NOTE: On day 1, the culture may contain significant amounts of debris due to the residue myelin from the spinal cord tissue; thus, changing the media on day 1 is recommended. Cultures are ready for treatment between D 12 – 14. Cells usually are 80% confluent at D 12 and can be near 100% confluent by D 14. Typically, on D 12, there are about 100,000 cells per well in a 12-well plate.