Building Three-Dimensional Neuronal Networks Coupled to Micro-Electrode Arrays

Abstract

Source: Tedesco, M., et al., Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model. J. Vis. Exp. (2015)

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

1. 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.
2. Anesthetize a pregnant female rat after 18 days of development (E18) using 3% isoflurane. Then, sacrifice the animal by cervical dislocation.
3. Remove hippocampi from each rat embry....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 w\o 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 8x8; 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)
HBSS w\o calcium and magnesium ions	Gibco Invitrogen	14175-052
Hanks Buffer Solution	Sigma	H8264
Rat	Sprague Dawley	Wistar Rat