Regulating Schwann Cell Growth In Vitro Using the Nanosecond Pulsed Electric Field Technique

Abstract

Source: Han, J., et.al. Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro. J. Vis. Exp. (2024)

This video demonstrates the application of the nanosecond pulsed electric field (nsPEF) technique to modulate Schwann cell (SC) growth. The rapid electric pulses induce transient nanopores in SC membranes, facilitating calcium influx that activates signaling pathways driving SC proliferation, dedifferentiation, and neurotrophic factor secretion. These cellular responses enhance axonal support, neuronal survival, and potential myelination, highlighting nsPEF as a promising tool for nerve repair.

Protocol

1. Thawing of cryopreserved RSC96 cells

1. Thaw the cryovial containing 1 mL of cell suspension by rapidly shaking it in a 37 °C water bath and then add it to a centrifuge tube containing 4-6 mL of complete culture medium and mix well.
2. Centrifuge at 1000 x g for 3-5 min, discard the supernatant and resuspend the cells in 3 mL of complete culture medium.
3. Add the cell suspension to a culture flask (or dish) containing 6-8 mL of complete culture medium and incubate at 37 °C overnight.
4. The next day, observe the cell growth and density under a microscope.

2. Cell passage:

NOTE: If the cell density reaches 80%-90%, it is ready for passage.

1. Discard the culture medium and rinse the cells 1-2 times with phosphate-buffered saline (PBS) without calcium and magnesium ions.
2. Add 0.25% (w/v) trypsin-0.53 mM EDTA to the culture flask (1-2 mL for a T25 flask, 2-3 mL for a T75 flask) and incubate at 37 °C for 1-2 min.
3. Observe the cell detachment under a microscope. If most cells become round and detach, quickly return the flask to the working area, tap the flask gently, and add 3-4 mL of culture medium containing 10% FBS to stop the digestion.
4. Mix the contents, aspirate the solution, and centrifuge at 1000 x g for 5 min. Then, discard the supernatant and resuspend the cells by adding 1-2 mL of fresh culture medium and pipetting gently.
5. Transfer the cell suspension to a new T25 flask at a 1:2 ratio and add 7 mL of culture medium.

3. Operation of nsPEF device

1. Resuspend the RSC96 cells in 1 mL of DMEM culture medium and transfer them to colorimetric dishes with electrodes on both sides.
2. Turn on the power switch.
3. Adjust the parameters by rotating the knob on the instrument to alter the intensity of the electric field. The intensities set in this study are 5 kV/cm, 10 kV/cm, 20 kV/cm, and 40 kV/cm.
4. Carefully rotate the electrodes until sparks appear, allowing the cells to receive 5 pulses of nsPEF according to the preset field strength intensities before immediately separating the two electrodes.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
High Voltage Power Supply for nsPEF	Matsusada Precision Inc.	AU-60P1.6-L
Pipette	Wuhan Xavier Biotechnology Co., LTD
RSC96 Schwann cells	Wuhan Xavier Biotechnology Co., LTD	STCC30007G-1
Scanister	3DHISTECH	Pannoramic MIDI
Special cable for nsPEF	Times Microwave Systems	M17/78-RG217