1Institute for Biomaterials and Biomedical Engineering, University of Toronto, 2Lyndhurst Centre, Toronto Rehabilitation Institute, 3Department of Surgery, University of Toronto
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Babona-Pilipos, R., Popovic, M. R., Morshead, C. M. A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field. J. Vis. Exp. (68), e4193, doi:10.3791/4193 (2012).
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.
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 galvanotaxis of undifferentiated NPCs because these mitogens maintain NPCs in their undifferentiated state4. When investigating the galvanotaxis of NPCs induced to differentiate into mature cell types, "FBS-SFM" refers to serum free media supplemented with 1% fetal bovine serum. FBS promotes the differentiation of NPCs into mature neural phenotypes5.
1. Isolation and Culture of Neural Precursors (Not shown in video)
2. Galvanotaxis Chamber Preparation
3. Live Cell Time-Lapse Imaging
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 absence or presence of a dcEF.
Differentiated phenotypes undergo negligible migration both in the presence and the absence of a dcEF (Movies 3, 4). Subsets of differentiated cells extend processes that tend to align perpendicular to the direction of the dcEF, but no noticeable cell body translocation is observed.
Immunostaining verifies that NPCs maintain positive expression of the neural precursor marker nestin after 6 hr of dcEF exposure (Figure 6A). In the assays involving NPCs induced to differentiate into mature phenotypes, the majority of cells express the mature astrocyte marker glial fibrillary acidic protein (GFAP) after 6 hr of dcEF exposure (Figure 6B).
The primary antibodies used in these analyses were as follows: mouse monoclonal anti-nestin (1:400, Millipore, Canada), and rabbit polyclonal anti-GFAP (1:500, Sigma, Canada). The secondary antibodies used in these analyses were as follows: goat-anti-mouse conjugated with Alexafluor 568 (1:400, Invitrogen-Gibco, Canada), and goat-anti-rabbit conjugated with Alexafluor 488 (1:400, Invitrogen-Gibco, Canada).
Figure 1. Neurospheres adhere to the Matrigel substrate and dissociate into single cells following 17 hr of incubation at 37 °C/5% CO2, 100% humidity in EFH-SFM.
Figure 2. Illustration of galvanotaxis chamber. Strips of vacuum grease are used to create a pool of culture media on either side of the central trough.
Figure 3. Schematic of holes that should be drilled into the lids the galvanotaxis chamber and the culture medium reservoirs. The 7 mm diameter holes in both lids are used to insert the agarose gel tubes. The 4 mm diameter holes in the lid of the galvanotaxis chamber is used to measure the electric potential directly across the central trough using a voltmeter. The 4 mm diameter hole in the lid of the culture medium reservoir is to allow the Ag/AgCl electrode to protrude from the lid of the dish for connection to the power supply.
Figure 4. Illustration of galvanotaxis chamber assembly for time-lapse imaging. The galvanotaxis chamber is the central Petri dish, in which the cells are plated. The media inside the central Petri dish housing the galvanotaxis chamber is either SFM + EFH or SFM + 1% FBS. The Petri dishes on either side of the galvanotaxis chamber are filled with SFM, and also contain the Ag/AgCl electrodes. These electrodes are connected to an external power supply, and bridging the three Petri dishes with agarose-gel bridges forms a complete circuit.
Figure 5. In the presence of a dcEF undifferentiated NPCs undergo rapid galvanotactic migration toward the cathode (A), whereas in the absence of a dcEF the cells undergo random radial migration (B).
Figure 6. Immunostaining verifies that NPCs remain nestin-positive undifferentiated precursors after 6 hr of dcEF exposure (A), and that NPCs induced to differentiate mostly express the mature astrocyte marker GFAP after 6 hr of dcEF exposure (B).
Movie 1. Time-lapse video of undifferentiated NPCs exposed to a dcEF. NPCs plated onto galvanotaxis chambers for 17 hr in SFM + EFH, and then exposed to a 250 mV/mm dcEF exhibit rapid and directed migration toward the cathode. 1 second of video = 15 minutes real time. Click here to view movie.
Movie 2. Time-lapse video of undifferentiated NPCs in the absence of a dcEF. NPCs plated onto galvanotaxis chambers for 17 hr in SFM + EFH, and then imaged in the absence of an applied dcEF undergo random migration. 1 second of video = 15 minutes real time. Click here to view movie.
Movie 3. Time-lapse video of differentiated NPCs exposed to a dcEF. NPCs plated onto galvanotaxis chambers for 69-72 hr in SFM + FBS, and then exposed to a 250 mV/mm dcEF exhibit very little migration in any direction. 1 second of video = 15 minutes real time. Click here to view movie.
Movie 4. Time-lapse video of differentiated NPCs in the absence to a dcEF. NPCs plated onto galvanotaxis chambers for 69-72 hr in SFM + FBS, and then imaged in the absence of an applied dcEF exhibit very little migration in any direction, similar to differentiated NPCs that are exposed to a dcEF. 1 second of video = 15 minutes real time. Click here to view movie.
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 using materials commonly found in most cell biology laboratories. Our protocol includes a heated, humidified, CO2 regulated incubator that surrounds the live cell imaging system to maintain the cells under optimal conditions for continuous long-term imaging. The lack of such apparatus is a limitation of some previously published techniques9,12.
Recent work by another group demonstrated similar galvanotactic behavior of cells from a hippocampal cell line13, using a different protocol and a more labor-intensive galvanotaxis chamber design. Our assay is particularly elegant in that it permits an analysis of the identical starting population of cells - primary cell cultures of undifferentiated neurosphere derived NPCs - that have been exposed to distinct conditions thereby permitting a comparison of the migratory properties of both undifferentiated and differentiated cells, simply by modifying the factors used to supplement the culture media within the galvanotaxis chamber.
The assay presented in this protocol is a powerful tool for long-term tracking and analysis of NPC galvanotaxis, and possibly other cell types that undergo galvanotactic migration14,15. As with any protocol, nuances in the preparation and execution of certain steps in this protocol may lead to unsuccessful results. The following guidelines and suggestions should assist in the successful execution of this protocol:
Our lab routinely images NPC galvanotaxis for 6-8 hr, although up to 15-hr analyses have been performed. Over the 15-hr periods, the dcEF across the chamber declined from 250 mV/mm to 227 mV/mm (9.2% decrease). Longer imaging periods inevitably modify the pH of the media in the chamber, further modifying the electrical field. Thus it is recommended to replace the media approximately every 6 hr. However, we have performed 8-hr NPC galvanotaxis assays without media replacement and have not observed any noticeable cell death (dead cells do not migrate) or decline in cell motility; the cells maintain their velocity of migration throughout the experiment. The investigator may also reduce the depth of the chamber to increase the stability of the dcEF10.
Following imaging, single-cell kinematic tracking analysis is performed on a subset of the imaged cells using Zeiss Axiovision software's tracking module. Cells are selected for analysis if they are localized closer to the edge of the dissociated neurosphere and at least one cell body apart from its nearest cell. The rationale behind this is to minimize the likelihood of cells overlapping each other during tracking, which would make tracking individual cells near impossible. We analyze the following four parameters of migration:
The latter two parameters are indicators of how straight the migration pathway is in a particular direction.
Upon grasping the methods in this protocol, the investigator may wish to modify some aspects for broader applications. For example, the assay may also be modified to investigate the migratory behavior of other cell types. Genetic knockout models or siRNA transfection techniques could be used to target genes of interest that may play a role in cell migration or the transduction of a dcEF into cell motility. In our lab, we have modified the galvanotaxis chamber to permit continuous cross-perfusion of fresh culture media within the central trough during experimentation18. An appropriate substrate and culture media would need to be selected for other cell types, and the duration of cell seeding prior to imaging should be adjusted as necessary. Notably, the dimensions of the chamber may need to be modified depending on the thickness of the tissue being examined (for instance, if a tissue slice is placed in the chamber). The investigator should bear in mind that for a set electrical field, the current flow through the chamber is directly proportional to the cross-sectional area of the chamber, and therefore significant enlargements to the chamber's dimensions may result in increased Joule heating-related cell death.
The techniques described in this report provide a powerful tool for investigating the important, but not widely studied, phenomenon of galvanotaxis. The ability of cells to respond to dcEFs has direct therapeutic implications. Electrical stimulation (deep brain stimulation) has already proven useful in the treatment of Parkinson's disease, and has been shown to promote hippocampal neurogenesis in a mouse model19,20. A complete understanding of the cellular mechanisms responsible for dcEF transduction into cellular motility may eventually lead to the use of electrical stimulation as a clinical means for enhancing and directing endogenous precursor migration to sites of injury or disease to facilitate the repair process. The methods described above provide a simple and robust way of investigating these processes.
No conflicts of interest declared.
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.
|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|
|Bovine pancreas trypsin||Sigma||T1005|
|Sheep testes hyaluronidase||Sigma||H6254|
|Ovomucoid trypsin inhibitor||Worthington||LS003086|
|1M HEPES||Sigma||H3375||23.83 g dissolved in 100 ml dH2O|
|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.|
|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|
|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|
|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|
|Live Cell Time-Lapse Imaging|
|Silver wire||Alfa Aesar||11434|
|Heat Inactivated FBS||Sigma||16140071|
|PVC tubing||Fisher Scientific||80000006||3/32"ID x 5/32"OD|
|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|
Artificial cerebrospinal fluid
Trypsin Inhibitor Solution
Hormone Mix (100 ml total, store at -20 °C)
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
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.
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.