A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Summary

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

Abstract

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations¹. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF^2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

Protocol

All procedures involving animal handling were approved by the University of Toronto Animal Care Committee in accordance with institutional guidelines (protocol no. 20009387). The following methods should be performed using sterile tools and techniques, in a laminar flow hood where applicable.

In the protocol text below, the phrase "EFH-SFM" refers to serum free media supplemented with epidermal growth factor, basic fibroblast growth factor and heparin. EFH-SFM is used when investigating the ga.......

Discussion

This protocol has been adapted from the well-established methods of previous studies^7-9. Galvanotactic chambers can be constructed using a variety of different techniques, including the construction of a separate glass well for confinement of cell seeding, or using CO₂ laser ablation for microfabrication of the central trough^10,11. Some techniques may be more laborious or costly than others. We have described a simple and cost-effective assay for constructing a NPC galvanotaxis chamber us.......

Materials

Name	Company	Catalog Number	Comments
Name of item	Company	Catalogue number	Comments
			Neural Precursor Cell Isolation
2M NaCl	Sigma	S5886	11.688 g dissolved in 100 ml dH2O
1M KCl	Sigma	P5405	7.456 g dissolved in 100 ml dH2O
1M MgCl2	Sigma	M2393	20.33 g dissolved in 100 ml dH2O
155 mM NaHCO3	Sigma	S5761	1.302 g dissolved in 100 ml dH2O
0.5M Glucose	Sigma	G6152	9.01 g dissolved in 100 ml dH2O
108 mM CaCl2	Sigma	C7902	1.59 g dissolved in 100 ml dH2O
Penicillin-streptomycin	Gibco	15070
Bovine pancreas trypsin	Sigma	T1005
Sheep testes hyaluronidase	Sigma	H6254
Kynurenic acid	Sigma	K3375
Ovomucoid trypsin inhibitor	Worthington	LS003086
DMEM	Invitrogen	12100046
F12	Invitrogen	21700075
30% Glucose	Sigma	G6152
7.5% NaHCO3	Sigma	S5761
1M HEPES	Sigma	H3375	23.83 g dissolved in 100 ml dH2O
L-glutamine	Gibco	25030
EGF	Invitrogen	PMG8041	Reconstitute in 1 ml of hormone mix and aliquot into 20 μl units.
FGF	Invitrogen	PHG0226	Reconstitute in 0.5 ml of hormone mix and aliquot into 20 μl units.
Heparin	Sigma	H3149
Apo-transferrin	R&D Systems	3188-AT	0.1 g dissolved into 4 ml dH20
Putrescine	Sigma	P7505	Dissolve 9.61 mg into Apo-transferrin solution
Insulin	Sigma	I5500	Dissolve 25 mg into 0.5 ml of 0.1N HCl and add to 3.5 ml of dH20
Selenium	Sigma	S9133
Progesterone	Sigma	P6149
Standard Dissection Tools	Fine Science Tools
Dissection microscope	Zeiss	Stemi 2000
			Galvanotaxis Chamber Preparation
Square glass cover slides	VWR	16004
6N Hydrochloric Acid	VWR	BDH3204-1
High vacuum grease	Dow Corning
60 mm Petri dishes	Fisher Scientific	0875713A
Poly-L-lysine	Sigma	P4707
Matrigel	BD Biosciences	354234	Thaw and aliquot into 150 μl units
FBS	Invitrogen	10082139	Only use if inducing NPC differentiation, otherwise use SFM + EFH culture media as indicated above
Counting microscope	Olympus	CKX41
			Live Cell Time-Lapse Imaging
Silver wire	Alfa Aesar	11434
UltraPure Agarose	Invitrogen	15510-027
Heat Inactivated FBS	Sigma	16140071
PVC tubing	Fisher Scientific	80000006	3/32"ID x 5/32"OD
Bleach	Clorox
10 cc syringe	BD	309604
18 gauge needle	BD	305195
Dremel drill	Dremel	Model 750
Inverted microscope equipped with humidified, incubated chamber	Zeiss	Axiovert-200M
			Recipes Item Volume 2M NaCl 6.2 ml 1M KCl 0.5 ml 1M MgCl2 0.32 ml 155mM NaHCO3 16.9 ml 1M Glucose 1 ml 108 mM CaCl2 0.09256 ml Penicillin-streptomycin 1 ml Autoclaved water 74 ml Artificial cerebrospinal fluid Item Volume or Mass Artificial cerebrospinal fluid 30 ml Bovine pancreas trypsin 40 mg Sheep testes hyaluronidase 22.8 mg Kynurenic acid 5 mg Trypsin Solution Item Volume or Mass SFM 15 ml Ovomucoid trypsin inhibitor 10 mg Trypsin Inhibitor Solution Item Volume Autoclaved water 37 ml 10X DMEM/F12 10 ml 30% Glucose 2 ml 7.5% NaHCO3 1.5 ml 1M HEPES 0.5 ml Transferrin, Putrescine solution 4 ml 25 mg insulin solution 4 ml Selenium 100 μl Progesterone 100 μl Hormone Mix (100 ml total, store at -20 °C) Item Volume Autoclaved water 37.5 ml 10X DMEM/F12 (3:1) 5 ml 30% Glucose 1 ml 7.5% NaHCO3 0.75 ml 1M HEPES 0.25 ml Hormone mix 5 ml L-glutamine 0.5 ml Penicillin-streptomycin 0.5 ml Serum Free Media EFH-SFM: add 10 μl of EGF, 10 μl of FGF, and 3.66 μl of Heparin FBS-SFM: add 0.5 ml FBS Item Volume Matrigel 150 μl SFM 3.6 ml Matrigel Solution Matrigel aliquot should be placed in a box of ice and allowed to thaw slowly over 4-5 hours to form a viscous liquid before mixing with SFM. This will ensure the formation of a smooth layer of Matrigel substrate. If not thawed slowly, the resulting substrate will contain clumps of Matrigel, possibly hindering cell migration. Item Volume or Mass UltraPure Agarose 300 mg in 10 ml ddH20 SFM Heat Inactivated FBS 8 ml 2 ml Matrigel Solution Mix 8 ml of SFM with 2 ml heat inactivated FBS in a 15 cc falcon tube. Mix agarose with 10 ml ddH20 in an Erlenmeyer flask, and heat in a microwave for 30 sec in 10-sec intervals, ensuring to remove the solution from the microwave after each 10-sec interval and thoroughly mix. Following the final 10-sec microwave period, mix the agarose solution with the SFM/FBS solution and store in a 57 °C water bath.