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

Podsumowanie

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.

Streszczenie

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.

Protokół

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)

1. Anaesthetize a CD1 mouse (6-8 weeks old) with isofluorane and sacrifice via cervical dislocation.
2. Douse the head in 70% ethanol and decapitate the animal with sharp dissection scissors.
3. While holding the head with surgical forceps, remove the skin on the dorsal surface to expose the skull.
4. Using a scalpel and no. 11 blade, score the skull at the frontal sinus along the mediolateral axis, and also along the sagittal suture in the rostrocaudal direction.
5. Peel the parietal bones away from the head with no. 7 curved forceps, taking care not to pierce the brain tissue.
6. Insert a thin spatula underneath the brain starting from beneath the cerebellum and advancing toward the olfactory bulbs. While holding the skull in place with forceps, gently pull the brain from the skull and immediately place it in ice-cold artificial cerebrospinal fluid (see recipes below).
7. Under a dissection microscope, using sterile dissection scissors and forceps cut the brain in half along the midline. Rotate each hemisphere so that the medial (cut) surface faces upwards.
8. Select a hemisphere, and with the medial surface facing upward, locate the splenium of the corpus callosum (posterior region of the corpus callosum).
9. Make an incision from the surface of the cortex to the splenium of the corpus callosum along the dorsoventral axis.
10. Peel the incised cortex toward the olfactory bulb to expose the medial and lateral walls of the lateral ventricle.
11. Rotate the hemisphere so that the dorsal surface faces upwards, and use curved microscissors to cut out and collect the exposed medial and lateral walls, which contain the periventricular region where NPCs reside⁶.
12. Repeat steps 1.8-1.11 for the other hemisphere.
13. Pipette the isolated tissue into 7 ml of trypsin solution (see recipe below) in a 15 cc tube, and place the tube on a rocker in a 37 °C incubator for 25 min.
14. Centrifuge the tube at 1,500 rpm for 5 min, aspirate the supernatant, and resuspend the tissue in 2 ml of trypsin inhibitor solution (see recipe below).
15. Gently triturate the tissue with a small borehole Pasteur pipette 30-50 times carefully to avoid air bubbles.
16. Centrifuge the tube at 1,500 rpm for 5 min, aspirate the supernatant, and resuspend in 1-2 ml of SFM (see recipe below) by triturating the pellet 3-5 times.
17. Centrifuge the tube at 1,500 rpm for 3 min, aspirate the supernatant and resuspend in 1 ml of SFM + EFH.
18. Count live cell density with a haemocytometer and plate the cells in a T25 culture flask at a density of 10 cells per μl in SFM + EFH.
19. Allow the culture to grow for 7 days undisturbed to yield free-floating primary neurospheres comprised of NPCs.

2. Galvanotaxis Chamber Preparation

1. Place 3 square glass no. 1 cover slips (22 x 22 x 0.17 mm) in a bottle of 6N hydrochloric acid overnight.
2. The next day, use a diamond-tip glass-cutter to cut 6 rectangular strips (22 x 5 x 0.17 mm) of glass from square no. 1 cover slips.
3. Transfer the acid-washed square slips and rectangular slips into a laminar flow hood. Wash the rectangular and square strips first with 70% ethanol, then with tissue culture-grade autoclaved water, and allow to dry on a Kim Wipe (for added sterility, the glass may be allowed to air dry).
4. Apply vacuum grease to the perimeter of one surface of the square glass slides, and seal them to the base of 60 mm plastic Petri dishes.
5. Apply vacuum grease along the long axis of one surface of the rectangular glass strips, and seal them to opposite edges of the square glass slides (such that they are parallel to each other) in order to create a central trough.
6. UV-sterilize the chambers for at least 15 min in the laminar flow hood.
7. Pipette 250-300 μl of poly-L-lysine onto the central trough of the chambers and incubate at room temperature for 2 hr.
8. Approximately 15 min prior to the end of the incubation period, prepare the Matrigel solution (see recipe below).
9. Aspirate the poly-L-lysine, wash the central troughs with 1 ml of autoclaved water, and pipette 250-300 μl of Matrigel solution onto the central troughs.
10. Incubate the chambers at 37 °C for 1 hr.
11. Aspirate the Matrigel solution and gently wash the central troughs with 1-2 ml of SFM.
12. Pipette 100 μl of EFH-SFM or FBS-SFM onto the central troughs and transfer the galvanotaxis chambers onto the stage of a counting microscope.
13. Pipette 3-4 ml of the neurosphere-containing culture into a 60 mm Petri dish and transfer the Petri dish to the stage of the counting microscope.
14. At a viewing objective of 5x, use a P10 pipette to transfer 5-8 whole neurospheres (up to four at a time) onto the central trough of each galvanotaxis chamber without dissociating them, and carefully spread the neurospheres around the central trough without disrupting the Matrigel substrate.
15. Add an additional 150-200 μl of EFH-SFM or FBS-SFM onto the central troughs.
16. Transfer the galvanotaxis chambers into a 37 °C, 5% CO₂, 100% humidified incubator for 17-20 hr (if analyzing undifferentiated NPCs) to allow the neurospheres to adhere to the Matrigel substrate and dissociate into single cells as shown in Figure 1. If analyzing differentiated NPCs, the incubation period should be extended to 69-72 hr to allow the differentiation of the cells.

3. Live Cell Time-Lapse Imaging

1. Allow the live cell imaging system to equilibrate at 37 °C, 5% CO₂ for a minimum of 30 min prior to initiation of the time-lapse recording.
2. Cut two 12 cm pieces of 1 mm diameter Silver wire, coil them from one end, and place them in Clorox bleach for 20 min to form Ag/AgCl electrodes.
3. Transfer the galvanotaxis chambers onto the stage of a counting microscope and select which chamber will be used for live-cell imaging migration analysis based on the following criteria: i) the neurospheres should be almost completely dissociated into single cells and ii) the cells should possess round morphologies with little-to-no processes extending from the cell bodies.
4. Transfer the selected galvanotaxis chamber into a laminar flow hood, along with a separate square no. 1 glass cover slip and vacuum grease.
5. Wash the cover slip first with 70% ethanol, then with autoclaved water, and apply a strip of vacuum grease on two parallel edges of the cover slip.
6. Aspirate the culture media from the central trough of the chamber, then quickly place the cover slip (grease-side facing down) onto the chamber such that the grease strips rest on the two parallel rectangular glass strips, effectively creating a roof to the chamber.
7. Pipette 100 μl of fresh EFH-SFM or FBS-SFM into the central trough via capillary action.
8. Use vacuum grease to create borders for pools of culture media on each end of the central trough, as shown in Figure 2.
9. Cut two 15 cm pieces of PVC tubing, and use a 10 cc syringe with an 18 gauge needle to carefully inject agarose solution into the tubing, ensuring no bubbles form in the tubes, and allow the gel to solidify for 5 min.
10. Transfer the galvanotaxis chamber to the live cell imaging system, along with the agarose gel tubes, Ag/AgCl electrodes, and a pair of empty 60 mm Petri dishes that will be used as culture media reservoirs and will contain the Ag/AgCl electrodes. Allow the galvanotaxis chamber to rest within the 37 °C, 5% CO₂ environment for 20-30 min.
11. During this time, prepare the lids of the 2 empty Petri dishes and the lid of the galvanotaxis chamber's Petri dish by drilling holes into them with a Dremel or similar tool as shown in Figure 3.
12. Pipette 1-1.5 ml of EFH-SFM or FBS-SFM onto either side of the central trough, and 7-8 ml of SFM into each empty Petri dish. Place one Petri dish on each side of the galvanotaxis chamber's central trough and place one Ag/AgCl electrode into each dish. Bridge the gap between the galvanotaxis chamber and the Petri dishes to establish electrical continuity with the agarose gel bridges, as shown in Figure 4.
13. Connect the Ag/AgCl electrodes to an external power supply, with an ammeter in series to measure electrical current, and turn on the power supply. Use a voltmeter to measure the strength of the electric field directly across the central trough, and adjust the output of the power supply until the desired electric field strength is achieved (the assays performed in this lab utilize a dcEF strength of 250 mV/mm with electrical current between 1 and 1.5 mA).
14. Initiate the time-lapse module on the live cell imaging system, and allow the experiment to run for the desired amount of time. After completion of the assay, fix the cells in 4% paraformaldehyde for standard immunostaining analysis.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog 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.