Dissociating and Culturing Neurons from Hippocampal Tissue Samples

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Dissection, Dissociation, and Culture of Hippocampal Neurons from Pregnant Mouse/Rat

1. Dissection and dissociation of hippocampal neurons for culture
   1. Thaw protease enzyme solution (3 mg/mL protease 23 in Slice dissection solution, SLDS, Table 1) and pre-warm plating media (Table 2) at 37 °C.
   2. Dissect the hippocampus and rinse with SLDS.
      NOTE: Four hippocampi provide enough cells for one 24-well or one 6-well plate.
   3. Remove SLDS, add 2 mL of protease enzyme solution, and incubate the sample at 37 °C, 5% CO₂, for 15 min.
   4. Wash the hippocampus explants with 5-10 mL of plating medium twice. Add 5 mL of plating medium. Dissociate hippocampal explants into single cells by generating a small vortex using a pipette with a 1-mL plastic tip. Pipette up and down about 40 times, avoiding the formation of oxygen bubbles, until the solution becomes cloudy and no large pieces of explant remain.
      NOTE: Remember to leave some media during washes, the hippocampal explants should remain submerged in the medium during the whole process.
   5. Centrifuge the cells at 1,125 x g for 3 min. Remove the supernatant with the cells submerged in the media. Resuspend the cells in 145 mL of plating media. Add 1 mL/well of the cell suspension to a 24-well plate (3 mL/well to a 6-well plate).    
      NOTE: 145 mL of plating media is used to produce a cell suspension that has a low cell density. This volume will allow the plating of cells into six 24-well plates, eight 6-well plates, or various combinations of 24- or 6-well plates, depending on the experiments to be conducted. Each well of the 24-well plate will have about 3 x 10⁴ cells.
   6. Incubate the cells in the plating media at 37 °C, 5% CO₂ for 2-4 h, then remove all plating media and replace it with fresh maintenance media.
   7. After 2 days (2 days in vitro, 2DIV), replace half of the media with 2 µM Ara-C (arabinosylcytosine) dissolved in maintenance media, which inhibits growth of fibroblasts, endothelial, and glial cells. After exposing cells to Ara-C for two days, replace the media with fresh maintenance media.

Table 1: Coverslip coating solution. Provides the recipe for preparing the coverslip coating solution used to adhere cultured cells to the coverslips.

Table 2: Cell culture media recipes. Provides the recipes for preparing media utilized in the culturing of primary hippocampal neuron cells.