All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Preparing glia cell feeder dishes (2 weeks before culture day)
- Dissect the cortex from a postnatal 1-day-old mouse brain and peel off the meninges.
- Chop the cortex tissue as finely as possible with clean scissors in a clean Petri dish on a clean bench.
- Transfer the chopped tissue into 12 mL of HBSS and add 1.5 mL of 2.5% trypsin and 1% (wt/vol) DNase. Incubate in a 37 °C water bath for 15 min, swing every 5 min. Well-digested tissue becomes sticky and forms a big cluster. Triturate 10-15 times with a 10 mL pipette to break the tissue down and get better digestion.
- Triturate the well-digested tissue 10-15 times with a 5 mL pipette till most chunks disappear and the medium turns cloudy. Pass through a cell strainer to remove remaining chunks and add 15 mL of glia medium (Minimal essential medium (MEM) supplemented with glucose (0.6% wt/vol), 10% (vol/vol) horse serum, and Penicillin-Streptomycin (1x) to stop the digestion.
- Centrifuge the cells at 120 x g for 5 minutes and aspirate the supernatant. Resuspend the cell pellet with fresh glia medium and seed in cell culture dishes (about 105 cells/cm2 ).
- Replace the medium with fresh glia medium the next day to remove unattached cells.
- Feed the glia dishes every 3-4 days with fresh glia medium. Slap the flask 5-10 times with a hand to dislodge loosely attached cells before changing medium.
- After 10 days of culture, glial cells should be nearly confluent. Detach glia cells with 0.25% trypsin-EDTA and seed about 105 cells in a new 60 mm cell culture dish. The remaining cells could be frozen for future use.
- 3 days before the culture day, change the glia medium to neuronal culture medium (Neurobasal-A Medium with 1x GlutaMAX-I and 1x B27 supplement)
2. Culture hippocampal neurons
NOTE: All steps are performed at room temperature.
- Dissect 6-8 hippocampi from postnatal 1-day-old pups from ANK3-E22/23f/f mice with HBSS medium in a Petri dish at room temperature. Chop the hippocampi with dissection scissors into smaller pieces. Transfer hippocampi from the Petri dish to a 15 mL tube.
- Wash hippocampi 2x with 5 mL of HBSS in the tube. Leave the hippocampi in 4.5 mL of 1x HBSS after washing.
- Add 0.5 mL of 2.5% trypsin into 4.5 mL of HBSS and incubate in a 37 °C water bath for 15 minutes. Invert the tube every 5 minutes. Well-digested hippocampi should become sticky and form a cluster. If needed, extend the digestion for 5 more minutes.
- Wash hippocampi with HBSS 3 times for 5 minutes each. Do not use a vacuum to remove the HBSS. It is very easy to remove the hippocampi.
- Add 2 mL of HBSS after the wash and pipette the hippocampi up and down with a Pasteur pipette 15 times.
- Triturate the tissue with a fire-polished Pasteur pipette (the diameter of the open is narrowed by half) 10 times. Do not go beyond 10 times even if there are still chunks remaining. Overshearing kills neurons.
- Rest the tube for 5 minutes till all chunks set to the bottom. Gently use a 1 mL pipette tip to transfer the supernatant containing the dissociated neurons to plating dishes (105 cells/60 mm dish). Add it directly to the preincubated plating medium and shake the plate gently.
- Repeat steps 2.6-2.7 with the remaining chunks till most of the chunks have disappeared.
- 2-4 hours after seeding, check the plating dishes with a light microscope. The majority of neurons should have attached to the coverslip. The attached cells are round and bright. Flip coverslips using a fine tip forceps to the glia cell feeder dishes with preconditioned neuronal culture medium with the wax dots side facing downwards.
- Neurons can grow in the glia cell feeder dishes for up to 1 month. Feed neurons every 7 days with 1 mL of fresh neuronal culture medium.
- Optional step: 1 week after seeding, add cytosine arabinoside (1-β-D-arabinofuranosylcytosine) to a final concentration of 5 μM to curb glial proliferation.
