Dissociating and Culturing Neurons from Hippocampal Tissue Samples

Samenvatting

Source: Servello, D. et al., A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons. J. Vis. Exp. (2018).

This video demonstrates the dissociation and culture of hippocampal neurons from hippocampal tissue samples using enzymatic and mechanical dissociations. The treatment with Ara-C inhibits the growth of non-neuronal cells, facilitating the selective growth of hippocampal neurons.

Protocol

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.

Coverslip coating solution
780 µL	Acetic acid17 mM stock: 50 µL acetic acid in 50 mL of H2O
200 µL	Poly-D-lysine (0.5 mg/mL stock)
20 µL	Rat tail collagen (3 mg/mL stock)
The total volume is determined based on the number of coverslips to be coated.

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

1x Slice dissection solution (SLDS), filtered, pH= 7.4, store at 4 °C
1 L	ddH2O
82 mM	Na2SO4
30 mM	K2SO4
10 mM	HEPES (free acid)
10 mM	D-glucose
5 mM	MgCl2
0.00%	Phenol red (optional)
Plating Media (PM, filter, store at 4 °C)
439 mL	MEM Earle's Salts
50 mL	FBS
11.25 mL	20% D-glucose
5 mL	Sodium pyruvate (100 mM,)
62.5 µL	L-glutamine (200 mM)
5 mL	Penicillin/Streptomycin (P/S, 100x)
Maintenance Media (MM, filter, store at 4 °C)
484 mL	Neurobasal
10 mL	B27 50x supplement
1.25 mL	L-glutamine (200 mM)
5 mL	100x P/S
All solutions are sterilized using a filter with 0.2 mm pore size.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
12 mm coverslips	Warner Instruments	64-0702	For 24-well plate
25 mm coverslips	Fisher Scientific	12-545-102	For 6-well plate
Acetic acid	Fisher Scientific	A38-212
Poly-D-lysine	Sigma	P6407
Rat tail collagen	Roche	11 179 179 001
10X PBS	National Diagnostics	CL-253
Na2SO4	Fisher Scientific	S373-500
K2SO4	Fisher Scientific	P304-500
HEPES	Fisher Scientific	BP410-500
D-glucose	Fisher Scientific	D16-500
MgCl2	Fisher Scientific	BP214-500
NaOH	Fisher Scientific	SS255-1
Protease enzyme	Sigma	P4032
FBS	Gibco	26140
Sodium pyruvate	Gibco	11360-070
L-glutamine	Gibco	25030081
Penicillin/Streptomycin 100x (P/S)	Gibco	15140122
MEM Earle's Salts	Gibco	11090
B27 supplement	Gibco	17504-044
Neurobasal	Gibco	21103-049
Arabinosylcytosine (Ara-C)	Sigma	147-94-4
Opti-MEM media	Gibco	31985-070