Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Summary

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Abstract

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system^1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans^3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types^5,6, including neural progenitor cells (NPCs)^7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously^5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures^9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

Protocol

1. Neural progenitor cell isolation

1. Dissect whole brains from E14-16 mice and place in cold DMEM/F12 basal medium. Remove all meninges under an anatomical microscope and transfer brains into a 35 mm Petri dish.
2. Use fine forceps to mechanically dissociate brains into tissue fragments and transfer them to a 15 ml tube, then centrifuge samples at 800 rpm for 3 min to remove debris.
3. Add DMEM/F12 containing bFGF and EGF and triturate with a 1 ml pipette.
4. Pass the cell suspension through a cell strainer to obtain a single cell suspension.
5. Plate cells into flasks at 2-5 x 10⁴ cells/ml, then perform a full medium ....

Discussion

The protocols we use are based on previous studies^5,11. Using these methods, stable culture and electric current conditions can be maintained while applying an EF via agar bridges, Steinberg's solution, and Ag/AgCl electrodes, to cells or slices cultured in custom-designed electrotactic chambers of standardised and precise dimensions. The depth of chambers can be adjusted to accommodate for different sample thicknesses¹¹, and in the case of cells, chamber size can be modified to accommodate however .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FGF-basic Recombinant Human	Invitrogen	PHG0026	20 ng/mL
EGF Recombinant Human	Invitrogen	PHG0311	20 ng/mL
N2-Supplement (100X) liquid	Invitrogen	02048
DMEM/F12 medium (high glucose)	Invitrogen	31330-095
Poly-D-Lysine	EMD Millipore	A-003-E
Natural mouse Laminin	Invitrogen	23017-015
Growth factor reduced Basement Membrane Matrix (Matrigel)	BD Biosciences	354230
HEPES buffer	GIBCO, by Life Technologies	15630
McIlwain tissue chopper	The Mickle Laboratory Engineering Co Ltd	TC752-PD
Dow Corning high-vacuum silicone grease	Sigma-Aldrich	Z273554