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In This Article

  • Summary
  • Abstract
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 dcEF2-3. 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.......

Representative Results

Kinematic analysis reveals that in the presence of a 250 mV/mm dcEF, undifferentiated NPCs exhibit highly directed and rapid galvanotaxis toward the cathode (Figure 5A, Movie 1). In the absence of a dcEF, random movement of the cells is observed (Figure 5B, Movie 2). At this field strength, > 98% of undifferentiated NPCs migrate for the entire 6-8 hr for which they are imaged, and since dead cells do not migrate this suggests that they remain viable during this period in the abs.......

Discussion

This protocol has been adapted from the well-established methods of previous studies7-9. 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 trough10,11. 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.......

Acknowledgements

This work is funded by the Natural Sciences and Engineering Research Council of Canada (grant #249669, and #482986) and Heart and Stroke Foundation of Canada (grant #485508). The authors thank Youssef El-Hayek and Dr. Qi Wan for their assistance in developing the experimental protocols.

....

Materials

NameCompanyCatalog NumberComments
Name of itemCompanyCatalogue numberComments
Neural Precursor Cell Isolation
2M NaClSigmaS588611.688 g dissolved in 100 ml dH2O
1M KClSigmaP54057.456 g dissolved in 100 ml dH2O
1M MgCl2SigmaM239320.33 g dissolved in 100 ml dH2O
155 mM NaHCO3SigmaS57611.302 g dissolved in 100 ml dH2O
0.5M GlucoseSigmaG61529.01 g dissolved in 100 ml dH2O
108 mM CaCl2 SigmaC79021.59 g dissolved in 100 ml dH2O
Penicillin-streptomycinGibco15070
Bovine pancreas trypsinSigmaT1005
Sheep testes hyaluronidaseSigmaH6254
Kynurenic acidSigmaK3375
Ovomucoid trypsin inhibitorWorthingtonLS003086
DMEM Invitrogen12100046
F12Invitrogen21700075
30% GlucoseSigmaG6152
7.5% NaHCO3SigmaS5761
1M HEPESSigmaH337523.83 g dissolved in 100 ml dH2O
L-glutamineGibco25030
EGFInvitrogenPMG8041Reconstitute in 1 ml of hormone mix and aliquot into 20 μl units.
FGFInvitrogenPHG0226Reconstitute in 0.5 ml of hormone mix and aliquot into 20 μl units.
HeparinSigmaH3149
Apo-transferrinR&D Systems3188-AT0.1 g dissolved into 4 ml dH20
PutrescineSigmaP7505Dissolve 9.61 mg into Apo-transferrin solution
InsulinSigmaI5500Dissolve 25 mg into 0.5 ml of 0.1N HCl and add to 3.5 ml of dH20
SeleniumSigmaS9133
ProgesteroneSigmaP6149
Standard Dissection ToolsFine Science Tools
Dissection microscopeZeissStemi 2000
Galvanotaxis Chamber Preparation
Square glass cover slidesVWR16004
6N Hydrochloric AcidVWRBDH3204-1
High vacuum greaseDow Corning
60 mm Petri dishesFisher Scientific0875713A
Poly-L-lysineSigmaP4707
MatrigelBD Biosciences354234Thaw and aliquot into 150 μl units
FBSInvitrogen10082139Only use if inducing NPC differentiation, otherwise use SFM + EFH culture media as indicated above
Counting microscopeOlympusCKX41
Live Cell Time-Lapse Imaging
Silver wireAlfa Aesar11434
UltraPure AgaroseInvitrogen15510-027
Heat Inactivated FBSSigma16140071
PVC tubingFisher Scientific800000063/32"ID x 5/32"OD
BleachClorox
10 cc syringeBD309604
18 gauge needleBD305195
Dremel drillDremelModel 750
Inverted microscope equipped with humidified, incubated chamberZeissAxiovert-200M

Recipes

ItemVolume
2M NaCl6.2 ml
1M KCl0.5 ml
1M MgCl20.32 ml
155mM NaHCO316.9 ml
1M Glucose1 ml
108 mM CaCl20.09256 ml
Penicillin-streptomycin1 ml
Autoclaved water74 ml

Artificial cerebrospinal fluid

ItemVolume or Mass
Artificial cerebrospinal fluid30 ml
Bovine pancreas trypsin40 mg
Sheep testes hyaluronidase22.8 mg
Kynurenic acid5 mg

Trypsin Solution

ItemVolume or Mass
SFM15 ml
Ovomucoid trypsin inhibitor10 mg

Trypsin Inhibitor Solution

ItemVolume
Autoclaved water37 ml
10X DMEM/F1210 ml
30% Glucose2 ml
7.5% NaHCO31.5 ml
1M HEPES0.5 ml
Transferrin, Putrescine solution4 ml
25 mg insulin solution4 ml
Selenium100 μl
Progesterone100 μl

Hormone Mix (100 ml total, store at -20 °C)

ItemVolume
Autoclaved water37.5 ml
10X DMEM/F12 (3:1)5 ml
30% Glucose1 ml
7.5% NaHCO30.75 ml
1M HEPES0.25 ml
Hormone mix5 ml
L-glutamine0.5 ml
Penicillin-streptomycin0.5 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

ItemVolume
Matrigel150 μl
SFM3.6 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.

ItemVolume or Mass
UltraPure Agarose300 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.

References

  1. Borgens, R. B., Shi, R. Uncoupling histogenesis from morphogenesis in the vertebrate embryo by collapse of the transneural tube potential. Dev. Dyn. 203, 456-467 (1995).
  2. Song, B., Zhao, M., Forrester, J. V., McCaig, C. D.

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