Dissociating and Culturing Neurons from Hippocampal Tissue Samples

Start with hippocampal tissue samples containing neurons and various non-neuronal cells.

Add a protease enzyme solution. The enzyme degrades the tissue’s extracellular matrix, initiating cell dissociation.

Wash with a plating medium to remove residual enzymes.

Pipette the digested tissue repeatedly to mechanically dissociate the cells from the tissue, forming a single-cell suspension.

Centrifuge and remove the supernatant. Resuspend the cells in the plating medium.

Seed the cells onto a poly-D-lysine-coated coverslip in a multi-well plate. Lysine enhances cell attachment.

Replace the plating medium with a neuron maintenance medium containing essential nutrients for neuron survival.

Refresh half of the media with maintenance media containing arabinosylcytosine, or Ara-C. 

Ara-C enters the cells and selectively inhibits the growth of non-neuronal cells, leading to their death.

Replace the media with fresh maintenance media to support neuron survival.

To dissociate the hippocampal neurons for culture, thaw the protease enzyme solution, and pre-warm the plating medium at 37 degrees Celsius. Next, rinse the dissected hippocampus with SLDS. Then remove it, and add 2 milliliters of protease enzyme solution, and incubate the sample at 37 degrees Celsius for 15 minutes.

Afterward, wash the hippocampus explants with 5 to 10 milliliters of plating medium twice, and add 5 milliliters of plating medium. Dissociate the hippocampal explants into single cells by generating a small vortex using a pipette with a 1-milliliter 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.

Next, centrifuge the cells at 1,125 times gravity for 3 minutes. Remove the supernatant with the cells submerged in medium, and resuspend the cells in 145 milliliters of plating medium. Then, add 1 milliliter of the cell suspension per well to a 24-well plate. Incubate the cells in the plating medium at 37 degrees Celsius for two to four hours.

Afterward, remove the plating medium and replace it with fresh maintenance medium. After two days, replace half of the medium with two micromolar Ara-C dissolved in maintenance medium to inhibit growth of fibroblasts, endothelial, and glial cells. Then, after exposing the cells to Ara-C for two days, replace the medium with fresh maintenance medium.