Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

1. Cell Cultures and Predifferentiation

1. Neural stem cell culture and predifferentiation
   1. Prepare poly-D-lysine-coated coverslips. Put a sterile coverslip (20 mm in diameter) into a 12-well plate. Coat the cover glass with poly-D-lysine, 0.1% w/v, in water (Table of Materials) for better cell adhesion on the coverslips by following the next steps.
      NOTE: Optimal conditions must be determined for each cell line and application.
      1. Aseptically coat the surface of the coverslip with poly-D-lysine, 0.1% w/v, in water. Rock gently to ensure an even coating of the coverslip surface.
      2. After culturing overnight (~12 h) at 37 °C, remove the poly-D-lysine solution and rinse the surface 3x with 1 mL of sterile water.
      3. Dry the cells at least 30 min before seeding them.
2. Murine neural stem cell C17.2 (C17.2-NSC) culture and predifferentiation
   1. Incubate C17.2-NSCs in a 25 cm² flask in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% horse serum (HS), and 1% penicillin/streptomycin at 37 °C and 5% CO₂ for ~2 – 3 d.
   2. When the cells reach 85% confluence, remove the medium, wash the cells with 1 mL of PBS and add 1 mL of fresh trypsin (0.25%) into the flask. Leave it for 1 min; then, add 1 mL of culture medium to the flask and pipette up and down several times to ensure a single cell suspension.
   3. Count the density of the cells in the suspension using a hemocytometer. Calculate the required volume of cell suspension to give a final cell concentration of 2 x 10⁴ cells/mL.
   4. Seed the C17.2-NSCs on the coated coverslips in the 12-well plate with the density of ~2 x 10⁴ cells per well. Check the cell density under a microscope.
      NOTE: For a better result, the optimal cell density must be determined before the immunofluorescence experiment.
   5. Incubate the cells at 37 °C in a cell incubator for 12 h to allow for attachment.
   6. After attachment, wash the cells 2x with 1 mL of PBS and cultivate the cells with differentiation medium consisting of DMEM/F12 with 1% N2 supplement for 48 h before the plasma treatment.
3. Primary rat neural stem cell culture and predifferentiation
   1. Culture the primary rat NSC suspension in rat NSC growth medium in uncoated T25 flasks at a density of 5 x 10⁵ cells.
      NOTE: The primary rat NSCs were isolated following the protocol of Xie et al.⁹.
   2. Check the formation of neurospheres under an inverted microscope. Make sure the morphology of the neurospheres exhibits spherical and transparent multicellular complexes.
   3. When the neurospheres reach a diameter of 3 mm or larger, transfer the neurosphere suspension to a 15 mL sterile centrifuge tube and let the neurospheres settle by gravity.
   4. Remove the supernatant carefully to leave the neurospheres in a minimal volume of medium.
   5. Rinse the neurospheres 1x with 5 mL of PBS and let the neurospheres settle by gravity. Remove the supernatant to leave a minimal volume of PBS.
   6. Add a suitable volume of differentiation medium to adjust the cell density to ~12 – 15 neurospheres per milliliter. The differentiation medium for primary rat NSCs consists of DMEM/F12, 2% B27, and 1% FBS.
   7. Seed 1 mL of neurosphere suspension onto each coated coverslip in the 12-well plate.
      NOTE: Shake the plate gently to ensure that the neurospheres are evenly distributed. This step is critical for a better immunofluorescence result.
   8. Place the plate into the incubator and allow it to predifferentiate for 24 h before the plasma treatment.

2. Preparation of the Plasma Jets

1. Choose a half-open quartz tube with an internal diameter of 2 mm and an external diameter of 4 mm. Insert a high-voltage wire with a diameter of 2 mm into the tube.
2. Insert the quartz tube with the high-voltage wire into a 5 mL syringe and use a holder to mount it inside the center of the syringe. Set the distance between the sealed end of the quartz tube and the syringe tip at 1 cm.
3. Connect a 1 m silicone rubber pipe (with an inner diameter of 12 mm) to the open end of the syringe and, then, connect it to the flowmeter and gas valve in sequence.

3. Acquisition of the Jets

1. Connect the circuit as shown in Figure 1. Connect the output wire of the power supply to the plasma jet device and, then, connect the tip of the high-voltage probe with the output wire to detect the voltage. Connect the other end of the high-voltage probe to the oscilloscope to record the information of the output voltage. Check the whole circuit and make sure the power supply, the oscilloscope, and the high-voltage probe are all grounded.
2. Check the gas line. Make sure the gas tube has been connected to the plasma jet device; then, open the gas valve of the helium (volume fraction, 99.999%) and oxygen (volume fraction, 99.999%) and set the gas flow to 1 L:0.01 L/min (He:O₂).
   NOTE: Let the gas flow for several minutes before turning on the power supply for the first time.
3. Set the pulse amplitude, frequency, and pulse width as 8 kV, 8 kHz, and 1600 ns, respectively. Check the circuit again and, then, turn on the output button to create a plasma jet.
   CAUTION: Do not touch the high-voltage wire at any time.

4. Plasma Treatment of Neural Stem Cell

1. Set the distance between the nozzle of the syringe and the cell well hole to 15 mm.
   NOTE: The distance is measured from the bottom of the platform where the 12-well plate is placed.
2. Take the 12-well plate out of the incubator and change the medium of the predifferentiated cells to 800 µL of fresh differentiation medium.
3. Divide the cells into three groups: an untreated control group, a 60 s He-and-O₂ (1%) gas flow treatment group, and a 60 s plasma treatment group.
   NOTE: There is no obvious liquid loss under the treatment condition.
4. Place the 12-well plate under the plasma jets, make sure the syringe nozzle is fixed in the center of each hole, and give the relevant treatment to the different groups mentioned in step 4.3.
   NOTE: The untreated controls were kept in differentiation medium at room temperature during the experimental procedure to ensure uniform treatment conditions. The He-and-O₂ (1%) gas flow treatment group was treated with only a He and O₂ (1%) gas flow, without plasma generation. All the treatments should be performed in triplicate.

5. Neural Stem Cell Differentiation

1. After treatment, remove the original culture and add 1 mL of new differentiation medium to each well.
2. Incubate the cells in the incubator at 37 °C and 5% CO₂ for 6 d. Change the medium every other day.
3. Check the differentiation status of the different groups daily under an inverted phase-contrast light microscope. Randomly select at least 12 fields and take photos to record the morphological changes.

6. Immunofluorescence Staining

1. Rinsing
   1. Take the samples out of the cell incubator and remove the medium by aspiration. Rinse the cells 1x with 1 mL of PBS.
      NOTE: After the differentiation, the cells are easily detached. It is necessary to add the PBS from the side of the culture wells to avoid washing off the cells.
2. Fixation
   1. Fix the cells with 500 µL of 4% paraformaldehyde for 20 min at room temperature. After the fixation, gently rinse the cells 3x with 1 mL of PBS, 5 min each, to remove the residual 4% paraformaldehyde.
      NOTE: The optimal time point for cell fixation must be determined according to the cell types. After the fixation, add 1 mL of PBS from the side of the culture wells and leave the sample on the table for 5 min. Do not use a lab shaker, to ensure a minimal loss of cells.
3. Permeabilization
   1. Permeabilize the samples with 0.2% TritonX-100 in PBS for 10 min at room temperature. After permeabilization, rinse the cells gently with 1 mL PBS for three times, 5 min each.
      NOTE: The optimal time point for cell permeabilization must be determined according to cell types. After permeabilization, add 1 mL PBS from the side of the culture wells, leave the sample on the table for 5 min. Do not use a lab shaker to ensure the minimal loss of cells.
4. Blocking
   1. Add 1 mL of 10% goat serum in PBS to each sample and leave the sample on the table for 1 h to block any nonspecific interactions.
      NOTE: Do not use a lab shaker, to prevent the cells from detaching from the slide. A rinse is not necessary in this step.
5. Incubation with primary antibody
   1. Dilute the primary antibody using primary antibody dilution buffer.
      NOTE: For optimal results, the final dilution ratio of the primary antibody must be determined by pretest. The dilution ratios of anti-Nestin (for undifferentiated stem cell), anti-β-Tubulin III (for neuron), anti-O4 (for oligodendrocyte), anti-NF200 (for mature neurons), anti-ChAT (for cholinergic neurons), anti-LHX3 (for motor neurons), anti-GABA (for GABAergic neurons), anti-serotonin (for serotonergic neurons), and anti-TH (for dopaminergic neurons) are 1:80, 1:200, 1:100, 1:100, 1:100, 1:200, 1:100, 1:200, and 1:100, respectively.
   2. Apply 300 µL of diluted primary antibodies to different samples.
   3. Incubate the cell samples overnight at 4 °C.
      NOTE: It is recommended to incubate primary antibodies at 4 °C to reduce the background and nonspecific staining.
   4. Remove the primary antibodies and gently rinse the cells 3x with 1 mL of PBS.
6. Incubation with secondary antibody
   1. Dilute the Cy3-conjugated or Alexa Fluor 488-conjugated secondary antibodies using 3% goat serum in PBS.
      NOTE: For optimal results, the final dilution ratio of the secondary antibody must be determined by pretest.
   2. Apply 300 µL of relevant secondary antibodies to detect each primary antibody.
      NOTE: All the subsequent steps need to be performed in the dark to prevent fluorescence quenching.
   3. Incubate the cell sample in the dark for 2 h at room temperature.
   4. Remove the secondary antibodies and rinse the cells gently with 1 mL of PBS for 10 min at room temperature.
7. Nuclear staining
   1. Apply 500 µL of Hoechst 33258 working solution to immerse the cell sample.
   2. Incubate the cell sample in the dark for 8 min at room temperature to label the nuclei.
   3. Gently rinse the cells 3x with 1 mL of PBS, 10 min each, protected from light.
      NOTE: For a minimal loss of cells, the optimal washing condition must be determined empirically.
8. Mounting
   1. Place one drop of mounting medium in the center of the microslide.
   2. Take out of the coverslips (with samples) using tweezers and carefully position the sample on top of the mounting medium. Avoid air bubbles.
   3. Remove any excess mounting medium with absorbent paper.
9. Fluorescence microscopy observation
   1. Observe the samples under the fluorescence microscopy equipped with filters for Hoechst 33258, Alexa Fluor 488, and Cy3. For each sample, randomly select at least 8 – 12 fields and record images with a camera.