A Technique to Culture Mouse Hippocampal Neurons

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)

1. Dissect the cortex from a postnatal 1-day-old mouse brain and peel off the meninges.
2. Chop the cortex tissue as finely as possible with clean scissors in a clean Petri dish on a clean bench.
3. 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.
4. 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.
5. 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 ).
6. Replace the medium with fresh glia medium the next day to remove unattached cells.
7. 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.
8. 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.
9. 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.

1. 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.
2. Wash hippocampi 2x with 5 mL of HBSS in the tube. Leave the hippocampi in 4.5 mL of 1x HBSS after washing.
3. 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.
4. 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.
5. Add 2 mL of HBSS after the wash and pipette the hippocampi up and down with a Pasteur pipette 15 times.
6. 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.
7. 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.
8. Repeat steps 2.6-2.7 with the remaining chunks till most of the chunks have disappeared.
9. 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.
10. 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.
11. Optional step: 1 week after seeding, add cytosine arabinoside (1-β-D-arabinofuranosylcytosine) to a final concentration of 5 μM to curb glial proliferation.