This video demonstrates the development of a 3D neuronal culture coupled to a planar microelectrode array with hippocampal neurons. The isolated neurons form a 2D network on the microelectrode array's active area and adhere to glass microbeads in a multi-well plate with a membrane insert. Upon transferring these neuron-coated microbeads to the electrode, they organize into an interconnected 3D neural network.
Protocol
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Dissection of Embryos and Dissociation of Tissue House adult female rats (200-250 g) at a constant temperature (22 ± 1 °C) and relative humidity (50%) under a regular light–dark schedule (light-on 7 AM–7 PM) in the animal facility. Ensure that food and water are freely available. Anesthetize a pregnant female rat a…
Divulgations
The authors have nothing to disclose.
Materials
Laminin
Sigma-Aldrich
L2020
0.05 µg/ml
Poly-D-lysine
Sigma-Aldrich
P6407
0.05 µg/ml
Trypsin
Gibco
25050-014
0.125% diluted 1:2 in HBSS wo calcium and magnesium ions
DNAase
Sigma-Aldrich
D5025
0.05% diluted in Hanks solution
Neurobasal
Gibco Invitrogen
21103049
Culture medium
B27
Gibco Invitrogen
17504044
2% medium supplent
Fetal bovine serum (FBS)
Sigma-Aldrich
F-2442
10%
Glutamax-I
Gibco
35050038
0.5 mM
gentamicin
Sigma-Aldrich
G.1272
5mg/liter
Poly-Dimethyl-Siloxane (PDMS)
Corning Sigma
481939
Curing agent and the polymer
Micro-Electrode Arrays
Multi Channel Systems (MCS)
60MEA200/30-Ti-pr
MEA with: Electrode grid 8×8; Electrode spacing and diameter 200 and 30 µm, respectively; plastic ring without thread
Microbead
Distrilab-Duke Scientific
9040
1gr Glass Part.Size Stds 40 µm
Transwell
Costar Sigma
CLS 3413
Multiwell plates with membrane insert (6.5 mm diameter porous 0.4 µm)