Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

All experimental procedures were performed in accordance with the French legislation and in compliance with the European Community Council Directive of November 24, 1986 (86/609/EEC) for the care and use of laboratory animals. The research on animals was authorized by the Direction Départementale des Services Vétérinaires des Bouches-du-Rhône and approved by the ethical committee of Provence Cote D'Azur (Apafis # 22689-2019100414103054).

1. Flexible device microfabrication (Figure 1)

1. Cleaning of the glass slides
   1. Sonicate glass slides in 2% soap solution for 15 min. Rinse them with water.
   2. Sonicate again in a mixture of 80% of pure acetone and 20% of pure isopropanol for 15 min.
      CAUTION: These solvents are harmful and flammable. Wear personal protective equipment (PPE) and handle them under a fume hood.
   3. Rinse the slides with isopropanol and dry with an air gun.
      NOTE: Ensure that acetone doesn't dry on the substrates during the whole process.
2. Gold electrode patterning (Figure 1A)
   1. Deposit a 3 µm layer of parylene-C (PaC) with a parylene deposition system (Figure 1Aa).
      1. Place the cleaned glass slides in the deposition chamber. Spray soap on the chiller, let it dry, and insert it into the designated cold trap of the deposition system. This anti-adhesive facilitates easy removal of PaC from the chiller after the deposition.
         CAUTION: PaC is an irritant and poses a health danger. Wear gloves while handling it.
      2. Weigh 6 g of PaC in an aluminum boat and place it in the furnace. Evacuate the machine (P = 10 mTorr) and start the deposition with the following parameters: T_Chiller = -100 °C, T_Furnace = 690 °C, T_Vaporizer = 175 °C, and T_Chamber = 135 °C.
      3. When the deposition is finished and the temperature of the vaporizer is below 40 °C, turn off the chiller, vaporizer, and furnace. Vent the machine and collect the samples.
   2. Activate the surface of the samples by oxygen plasma treatment for 30 s (100 W, 50 sccm).
   3. Spin-coat the plasma-treated samples with a negative photoresist at 1,000 x g for 40 s. Place the samples on a hot plate at 110 °C for 2 min (Figure 1Ab).
      CAUTION: The photoresist solution is flammable and causes irritation; wear PPE and handle it under a fume hood.
   4. Place an i-line filter in the beamline of the UV broadband contact aligner and expose the photoresist through a mask that features the interdigitated electrode design (Figure 1Ac).
      NOTE: Interdigitated electrodes with a gap of 50 or 250 µm were designed using a layout editor and photomasks were ordered from a company that produces polyester photomasks by laser photoplotting.
   5. Bake the above samples at 110 °C on a hot plate for 3 min and let them cool down to room temperature for 5 min. Immerse samples in a metal-ion-free developer for 3 min to remove the non-exposed photoresist. Rinse samples with water and dry them with an air gun (Figure 1Ad).
      CAUTION: The developer solution is an irritant; wear PPE and handle under a fume hood.
   6. Activate the surface of the samples by oxygen plasma treatment for 60 s (100 W, 50 sccm).
   7. Deposit a 20 nm adhesion layer of chromium and a 300 nm layer of gold with a thermal evaporator as follows (Figure 1Ae).
   8. Vent the evaporator machine and clip the samples (facing down) on the upper round plate with metal screws. Fill the dedicated crucibles, respectively, with chromium and gold. Seal and evacuate the machine to reach a pressure below 5·10^-6 Torr. Start the rotation of the sample holder.
   9. Select the crucible containing chromium and slowly increase the current going through it until a deposition rate of 0.2 Å·s^-1 is reached. Open the shutter and wait until 20 nm of chromium is deposited. Close the shutter and slowly ramp down the current until 0 mA.
   10. Select the crucible containing gold and slowly increase the current going through it until a deposition rate of 0.2 Å·s^-1 is reached. Open the shutter to evaporate gold, wait until 10 nm of gold is deposited, and then increase the deposition rate to 1.5 Å·s^-1 until approximately 300 nm is deposited. Close the shutter and slowly ramp down the current to 0 mA.
   11. Let the samples cool down to room temperature for 15 min after deposition. Stop the rotation of the sample holder, vent the machine, and collect the samples.
   12. Immerse the samples in a beaker with acetone. Place the beaker on a shaking plate set at 110 rpm for 15 min to lift off the photoresist. Rinse the samples with isopropanol and dry them with an air gun (Figure 1Af).
   13. Activate the surface of the samples by oxygen plasma treatment for 30 s (100 W, 50 sccm).
   14. Deposit a 3 µm insulation layer of PaC with a parylene deposition system (see step 1.2.1) (Figure 1Ag).
3. Outline opening (Figure 1B)
   1. Spin-coat the samples with a positive photoresist at 600 x g for 35 s. Place it on a hot plate at 110 °C for 2 min (Figure 1Ba).
      CAUTION: The photoresist solution is flammable and causes irritation; wear PPE and handle under a fume hood.
   2. Make sure there is no i-line filter in the beamline of the UV broadband contact aligner and expose the photoresist through a mask that features the outline of the device with a UV broadband contact aligner (Figure 1Bb).
   3. Immerse the samples in a metal-ion-free developer for 4 min to remove the exposed photoresist. Rinse the samples with water and dry them with an air gun (Figure 1Bc).
   4. Etch the outline through the two layers of PaC with a reactive ion etcher (160 W, 22 min, O₂: 50 sccm, CF₄: 10 sccm) (Figure 1Bd).
   5. Remove the remaining photoresist with acetone, rinse with isopropanol, and dry the samples with an air gun.
4. PEDOT:PSS coating (Figure 1C)
   1. Spin-coat a 2% soap solution at 70 x g for 35 s (Figure 1Ca).
   2. Deposit a 3 µm sacrificial layer of PaC with a parylene deposition system (see step 1.2.1) (Figure 1Cb).
   3. Spin-coat positive photoresist at 600 x g for 35 s. Place the samples on a hot plate at 110 °C for 2 min (Figure 1Cc).
   4. Make sure there is no i-line filter in the beamline of the UV broadband contact aligner and expose the photoresist through a mask that features the active surface of the electrodes (Figure 1Cd).
   5. Immerse the samples in a metal-ion-free developer for 4 min to remove the exposed photoresist. Rinse the samples with water and dry them with an air gun (Figure 1Ce).
   6. Etch the PaC with a reactive ion etcher to open the active surface of the electrodes (160 W, 24 min, O₂: 50 sccm, CF₄: 10 sccm). Check with a microscope that there is no residual PaC on the active surface (Figure 1Cf).
   7. Remove the remaining photoresist with acetone, rinse with isopropanol and dry the samples with an air gun.
   8. Activate the surface of the samples using oxygen plasma treatment for 90 s (100 W, 50 sccm).
   9. Mix a commercial dispersion of chemically polymerized PEDOT:PSS with 5 vol% of ethylene glycol (EG), and 0.1 vol% of dodecylbenzene sulfonic acid (DBSA). Sonicate for 15 min. Add 1 wt% of (3-glycydyloxypropyl) trimethylsiloxane (GOPS) and sonicate for 5 min. Filter the solution through a 1.2 µm filter.
      CAUTION: EG is an irritant and poses a health danger. DBSA is an irritant and corrosive. GOPS is corrosive. Wear appropriate PPE and handle these chemicals under a fume hood.
      NOTE: The total volume depends on the number of samples. For 10 standard glass slides, prepare at least 20 mL that would correspond to the following quantities: 18.78 mL of PEDOT:PSS, 1 mL of EG, 20 µL of DBSA, and 200 µL of GOPS.
   10. Spin-coat four layers of PEDOT:PSS solution at 150 x g for 35 s. After deposition of each layer, bake the samples at 110 °C for 60 s on a hot plate and cool them down to room temperature for 5 min before spinning the next layer (Figure 1Cg).
   11. Remove the sacrificial PaC layer by immersing the samples in water (Figure 1Ch).
   12. Bake the samples at 140 °C for 1 h.
   13. Immerse the samples in deionized water for 30 min to remove the remaining soap and low-molecular-weight compounds in the PEDOT:PSS film and to detach samples from the glass substrate (Figure 1Ci).
5. Connections and packaging (Figure 1D)
   1. Deposit a thin layer of acrylic paint on a polyimide film that is coated with copper (Figure 1Da). Use an aerosol to obtain a homogeneous layer of paint (Figure 1Db).
   2. Pattern the acrylic paint with a laser (75 kHz, 7 W, 1 laser pass, 400 mm·s^-1) to obtain two rectangular contact pads (5 mm x 15 mm; 1.5 mm gap) (Figure 1Dc).
   3. Wet etch the copper with saturated 30% (w/v) ferric chloride (FeCl₃) in water for 15 min at 40 °C (Figure 1Dd).
      CAUTION: FeCl₃ is an irritant and corrosive; handle it with gloves under a fume hood.
   4. Strip the acrylic paint with acetone by slightly rubbing it with a cloth (Figure 1De).
   5. Cut the patterned polyimide film into rectangular shapes (15 mm x 30 mm) with a laser (15 kHz, 10 W, 30 laser passes, 130 mm·s^-1) (Figure 1Df).
   6. Dispense silver paste with a three-axis dispensing machine at a pressure of three bar with a 330 µm diameter needle (5 m·min^-1) (Figure 1Dg).
      CAUTION: Silver paste is an irritant; handle with gloves.
   7. Align and attach the PaC probe with the polyimide film under a binocular microscope using tweezers (Figure 1Dh).
      NOTE: Alignment marks can be patterned in step 1.5.2 to facilitate the positioning of the probe on the contact pads.
   8. Bake at 140 °C for 2 h in the oven.
   9. Place a 1 cm² polyimide protection tape on the contact pads (Figure 1Di).
   10. Dip the interface where the PaC probe and polyimide film are connected in PDMS (Figure 1Dj).
   11. Bake it for 2 h at 50 °C.
   12. Remove the protection tape to open the contact pads (Figure 1Dk).
       NOTE: Microfabrication of in vitro devices is similar but steps 1.2.1, 1.3, and 1.5must be skipped.

2. Generation of Glioblastoma GCaMP6f stable cell line

1. Lentivirus production
   1. In a 75 cm² flask, culture a HEK 293T-derived cell line optimized for lentivirus production in 10 mL of Dulbecco's Modified Eagle's Medium (DMEM) containing 4.5 g·L^-1 of glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate and supplemented with 10% tetracycline-free fetal bovine serum (FBS), 100 units·mL^-1 of penicillin and 100 µg·mL^-1 of streptomycin for least for 3 days until 80% confluence.
   2. Remove the medium from the flask. Gently rinse the cells with 10 mL of Phosphate-Buffered Saline (PBS).
   3. Add 1 mL of 0.25% Trypsin/EDTA solution and incubate the flask for 5 min at 37 °C.
      CAUTION: Trypsin/EDTA solution poses a health danger; wear PPE and handle under a fume hood.
   4. Add 8 mL of culture medium. Gently flush the cell suspension.
   5. Count the cells and plate 4 x 10⁶ cells in a Petri dish in 8 mL of culture medium.
   6. The next day, dilute 25 µg of the plasmid containing the gene GCaMP6f and a selection marker conferring resistance to puromycin in a total volume of 600 µL of water. Add it to a tube of transfection reagent. Vortex for 10 s at 3,000 rpm and incubate the tube at room temperature for 10 min to allow the production of nanoparticles.
   7. Add the contents of the tube dropwise on the culture of HEK 293 T cells and rock gently by hand. Incubate cells at 37 °C for at least 4 h.
   8. Replace the media containing nanoparticle complexes with fresh media and return the cells at 37 °C.
   9. Three days later, collect the supernatant and centrifuge at 500 x g for 10 min to remove cellular debris. Collect the liquid phase containing viral particles.
      NOTE: The virus production in the supernatant can be confirmed using a quantitative lentiviral titer test and can be stored at -80 °C for at least 2 years.
2. Glioblastoma cells transduction
   1. In a 75 cm² flask, culture glioblastoma cells in 10 mL of DMEM containing 1 g·L^-1 of glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate and supplemented with 10% tetracycline-free FBS, 100 units·mL^-1 of penicillin, and 100 µg·mL^-1 of streptomycin for at least 4 days.
   2. Discard the medium and add the supernatant obtained in step 2.1.9 on the target cells.
   3. Add 5 µg·mL^-1 of Hexadimethrine Bromide in the medium to improve transduction. Incubate for 6 h at 37 °C. Replace the medium with 10 mL of fresh medium.
      CAUTION: Hexadimethrine Bromide is an irritant. Handle it with gloves.
3. Generation of a stable cell line
   1. Two to three days after transduction, add 10 mL of DMEM containing 1 g·L^-1 of glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, 10% FBS, 100 units·mL^-1 of penicillin, 100 µg·mL^-1 of streptomycin and supplemented with puromycin to kill the non-transduced cells. Culture cells in this media for at least 3 days.
      CAUTION: Puromycin is an irritant; handle it with gloves.
      NOTE: Sensitivity of cells to puromycin must be tested before transduction by culturing cells in their recommended medium containing different concentrations of puromycin. One day later, check the cells with a microscope. Choose the adequate concentration at which the majority of cells are dead but few are still alive, to ensure that the antibiotic is not too toxic and might kill the transfected cells as well.
   2. Remove the medium and rinse the cells with 10 mL of PBS.
   3. Add 1 mL of 0.25% of a Trypsin/EDTA solution and incubate the flask for 5 min at 37 °C.
   4. Add 8 mL of culture medium. Gently flush the cell suspension.
   5. Collect 100 µL of cell suspension and measure cell concentration with a cell counter. Aspirate 50 µL of cell suspension in a handheld automated cell counter with a 60 µm sensor.
   6. Seed 1 cell/well in a 96-well plate. For example, for a concentration of 1 x 10³ cells per mL, add 1 / (1 x 10³), i.e., 0.001 mL of cell suspension per well. Seed every well to increase the chances of success. Complete with culture medium to reach a total volume of 200 µL per well.
   7. One day later, find each well containing one cell and check its fluorescence (λ_exc = 490 nm and λ_em = 530 nm). Mark the wells containing only one transfected cell. Continue the growth for a few days until the well is almost confluent.
   8. Discard the medium and rinse the cells with 200 µL of PBS. Add 100 µL of 0.25% Trypsin/EDTA solution and incubate the 96-well plate for 5 min at 37 °C.
   9. Add 100 µL of medium and gently flush the cell suspension. Transfer the cell suspension in a Petri dish. Add 5 mL of medium and let the cells grow for a few days until the Petri dish is almost confluent.
   10. Discard the medium and rinse the cells with 5 mL of PBS. Add 1 mL of 0.25% Trypsin/EDTA solution and incubate the Petri dish for 5 min at 37 °C.
   11. Add 6 mL of medium and gently flush the cell suspension. Transfer the cell suspension to a T25 flask. Continue the growth for a few days until the flask is almost confluent.
   12. Discard the medium and rinse the cells with 5 mL of PBS. Add 1 mL of 0.25% Trypsin/EDTA solution and incubate the T25 flask for 5 min at 37 °C. Add 7 mL of DMEM containing 1 g·L^-1 of glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, and supplemented with 10% FBS, 100 units·mL^-1 of penicillin, and 100 µg·mL^-1 of streptomycin. Gently flush the cell suspension.
   13. Split the cell suspension to four T25 flasks (2 mL per flask) and add 5 mL of medium to each flask. Let the cells grow for a few days until the flasks are almost confluent.
   14. Repeat step 2.3.12 for three flasks and keep the last flask for step 3.1.3. Transfer the cell suspension in a 15 mL conical tube and centrifuge at 150 x g for 5 min. Discard the supernatant and resuspend the cell pellet in 900 µL. Gently mix the cells to maintain a homogeneous cell suspension.
   15. Transfer the cell suspension into cryogenic storage vials. Add 100 µL of dimethyl sulfoxide. Place the cryovials at -80 °C overnight. Transfer frozen cells to liquid nitrogen for further experiments.
       NOTE: The efficiency of the transfection can be assessed by adding 5 µM ionomycin calcium salt in the medium and checking the induced increase of fluorescence under a fluorescence microscope (λ_exc = 490 nm and λ_em = 530 nm).

3. 3D models

1. Spheroid culture
   1. Prepare a solution of 1% (w/v) agarose in deionized (DI) water.
      1. Add 100 g of agarose powder in 100 mL of DI water and heat the solution in a microwave oven until all the powder is dissolved. Stir the solution regularly to avoid clumps. Autoclave the solution for 20 min at 120 °C.
   2. Once retrieved from the autoclave, add 75 µL of agarose solution per well in a 96-well plate carefully. Deposit it on the side of the well to form a meniscus, resulting in a non-adherent round bottom. Let it solidify for 15 min at room temperature.
   3. Detach the cells (step 2.3.12) from the flask obtained in step 2.3.14.
   4. Add 10,000 cells per well of glioblastoma cells and complete to reach a total volume of 150 µL per well with DMEM containing 1 g·L^-1 of glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, and supplemented with 10% fetal bovine serum (FBS), 100 units·mL^-1 of penicillin and 100 µg·mL^-1 of streptomycin.
   5. Incubate cells at 37 °C without moving the plate for 3 days. Then, replace half of the media with fresh media every 2 days with a multi-channel pipette until further experiments. Keep the pipette tip in the upper part of the well to avoid damage to the agarose or the spheroid itself.
      NOTE: The size of the spheroids depends on the number of cells seeded and the cell line, so it must be adapted depending on the experiments.
2. The in ovo model
   1. Place fertilized eggs of Japanese quail (C. japonica) in an incubator (37 °C and 57% humidity) on trays with an automatic rotator that turns eggs every 2 h. This day is considered Embryonic Day (ED) 0.
   2. Wash plastic weighing boats by placing them in 70% (w/v) ethanol. Take out the weighing boats and dry them under a fume hood.
      NOTE: From this point, experiments are not performed in sterile conditions. However, clean conditions are required to avoid the development of mold on the embryos.
   3. On ED3, gently open the eggs using a tweezer with thin tips pre-washed with 70% (w/v) ethanol. Pour the embryo into a plastic weighing boat, cover it with another weighing boat and place it in a standard humidified incubator at 37 °C for 3 days.
   4. On ED6, make a small incision in the chorioallantoic membrane (CAM) with a 23 G needle.
   5. Using a pipette, place a 7-day-spheroid on the incision, and return the embryo to the incubator for 3 days, until further experiments.
      NOTE: A fluorescent dye can be injected into the eye of the embryo to visualize blood vessels.
   6. On the day of the experiment, place the flexible probe on top of the vascularized tumor using a micromanipulator.
3. The in vivo model
   NOTE: This part of the protocol is adapted from the one previously published in reference¹⁴. Adult multicolor fluorescent AMU-Neuroinflam mice (B6.Cg-Tg(Thy1-CFP)23Jrs(Ly6a-EGFP)G5Dzk(Itgax-EYFP)1Mnz/FD) were used; these mice present labeling of a subpopulation of Thy1⁺ neurons by transgenic expression of ECFP, labeling of peripheral LyzM⁺ inflammatory cells by transgenic expression of EGFP and labeling of a subtype of microglia expressing EYFP under the control of Cd11c⁺. In brief, the animals are lightly sedated with 1.5% isoflurane for 2 mins before any treatment or injection. Before the surgery, the animals are anesthetized with Ketamine (120 mg/kg; IP) and Xylazine (12 mg/kg; IP). Then, 3% Lidocaine gel is applied locally to alleviate any pain in the ears associated with the fixation of stereotactic support. Then, 0.25% Bupivacaine solution is administered to the surgical site to alleviate any pain due to the craniotomy. Once the mouse was prepared for surgery, a craniotomy of 4 mm diameter was performed according to reference¹⁴. With a 26 G needle, a hole was made in the dura-mater in the middle of the craniotomy and the tumor spheroid was injected with the injection system described in reference¹⁴. Additionally, as described here, a flexible electrode was placed on the GCamp6 or DsRed expressing tumor spheroid before sealing the craniotomy with a glass window. 
   1. Place a drop of Dulbecco's Phosphate-Buffered Saline (DPBS) so that it covers the craniotomy. Place the flexible electrode onto the drop of DPBS, and gently place the back of the probe with contact pads onto the mouse's back (Figure 4B).
      NOTE: Use sterile gloves and a “tip only” technique. Change the gloves if a non-sterile surface is contacted. Provide thermal support during this procedure. 
   2. Touch the DPBS drop with a small piece of paper to absorb DPBS until the probe can lay flat on the dura and follow the curvature of the brain. Ensure that a small layer of DPBS remains below the electrodes without escaping from the side of the electrode. This ensures a barrier against glue spillover during the next steps.
      NOTE: Sterilize all equipment before use.
   3. Place a small drop of silicone adhesive onto the probe and cover it with a 5 mm round cover glass. Push the cover glass down until the silicone is evenly distributed and the distance between the cover glass and the probe is minimal. Push the cover glass down for another 30 s so that silicone can solidify.
   4. To secure the cover glass, quickly apply superglue on its sides and push it down until the glue cures to solid.
   5. Using a toothpick, apply superglue at the probe's neck taking care that the superglue gets drawn under the neck to provide stable support for it.
   6. Cover the skull with dental cement to build a chronic cap. Take special care to cover the edges of the cover glass only.
   7. Lift the back of the probe and apply cement underneath the neck of the probe. Rest the probe onto the cement before it cures. Push down the neck of the probe gently with blunt forceps so that its surface is at the same level as that of the cover glass and not in the way of the microscope objective during the experiment.
   8. Cover the top of the probe neck with not more than 1.5 mm of dental cement layer to achieve a firm hold on the probe. Build a cement well presenting a 1.5 mm ridge at a distance of 1-2 mm around the cover glass to create a basin for the immersion fluid for the two-photon imaging (Figure 4C).
   9. After the cement has cured, apply buprenorphine post-surgical analgesics (0.05 mg/kg, 0.1 mL per 10 g of body weight subcutaneously) and maintain the animal in a warm atmosphere until it wakes up. This includes proximity to an infrared light bulb as well as wrapping the animal in a paper towel.
      NOTE: Place a thermometer at the level of the mouse to monitor the temperature.
   10. Characterize impedance in the 1-10 kHz range using a potentiostat.
   11. Let the animal recover from the surgery for at least 10 days. Administer anti-inflammatory drugs immediately after surgery and continue monitoring the animal's state to provide appropriate post-operative analgesia.

4. Pulsed Electric Field (PEF) delivery and imaging

1. Place the specimens under a fluorescence microscope. In the case of 3D models, tumors can only be observed from the top.
   NOTE: For the in ovo model, experiments were performed under an epifluorescence microscope (but is also possible with a two-photon microscope), whereas experiments on the in vivo model were performed under a two-photon microscope (Figure 6).
2. Connect a pulse generator to the contact pads of the devices, using pogo pin connectors (in vitro) or crocodile clips (in ovo and in vivo) (Figure 4A). Set the desired parameters (number of pulses, voltage, pulse duration, frequency) and apply PEFs by running the generator (Figure 4A). Measure fluorescence simultaneously to observe the effects of the PEFs in real-time.