This protocol allows the delivery of pulse electric fields by flexible electronics to study their therapeutic effects on glioblastoma. We do this by visualizing the tumor microenvironment in vivo with imaging. The bioelectronics is integrating into this protocol using several different models of increasing complexity in order to study the effect of pulsating fields on cancer.
The microfabrication techniques presented in this work can also be tested as a means of treating other types of cancers including patient-derived xenografts. This moves towards the idea of precision medicine tailored for each individual patient. These probes are made with standard microfabrication techniques, so the manufacturing is straightforward and anyone with a clean room can make them.
For gold electrode patterning, deposit a three micrometer layer of parylene C with a parylene deposition system by placing the clean glass slides in the deposition chamber. Weigh six grams of parylene C in an aluminum boat, place it in the furnace, evacuate the machine, and start the deposition with the parameters described in the manuscript. When the deposition is finished and the vaporizer temperature is below 40 degrees Celsius, turn off the chiller, vaporizer, furnace.
Vent the machine and collect the samples. Spin coat the plasma-treated samples with a negative photoresist at 1, 000 times G for 40 seconds. Expose the photoresist through a mask that features the interdigitated electrode design.
Then immerse samples in a metal ion-free developer for three minutes to remove the non-exposed photoresist. To deposit a 20 nanometer adhesion layer of chromium and a 300 nanometer layer of gold with a thermal evaporator, vent the evaporator machine and clip the samples on the upper round plate with metal screws. Fill the dedicated crucibles respectively with chromium and gold.
Seal and evacuate the machine and start the rotation of the sample holder. Select the crucible containing chromium and slowly increase the current until the deposition rate reaches 0.2 angstroms per second. Open the shutter, wait until the deposition of 20 nanometers of chromium, close the shutter, and slowly ramp down the current until zero milliamperes.
Select the gold containing crucible and slowly increase the current until a deposition rate of 0.2 angstroms per second. Open the shutter to evaporate the gold, wait until the deposition of 10 nanometers of gold, and then increase the deposition rate to 1.5 angstroms per second until approximately 300 nanometers are deposited. Close the shutter and slowly ramp down the current to zero milliamperes.
Immerse the samples in a beaker containing acetone. Then rinse the samples with isopropanol and dry them with an airgun. Spin coat four layers of PEDOT:PSS solution at 150 times G for 35 seconds.
Remove the sacrificial parylene C layer by immersing the samples in water. Immerse the samples in deionized water for 30 minutes to remove the remaining soap and low molecular weight compounds in the PEDOT:PSS film and detach the samples from the glass substrate. Add 75 microliters of molten 1%agarose solution in each well of the 96-well plate carefully and let it solidify for 15 minutes at room temperature.
Detach the transduced glioblastoma cells obtained during the stable cell line generation. Add 10, 000 glioblastoma cells per well and make up the total volume to 150 microliters per well using DMEM containing one gram per liter glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, 10%fetal bovine serum, 100 units per microliters of penicillin, and 100 micrograms per microliters of streptomycin. Replace half of the media with fresh media every two days until further experiments while keeping the pipette tip in the upper part of the well to avoid damage to the agarose or the spheroid itself.
Place the fertilized eggs of Japanese quail, Coturnix japonica, in an incubator on trays with an automatic rotator that turns eggs every two hours. This day is considered embryonic day zero. On embryonic day three, gently open the eggs using a tweezer with thin tips pre-washed with 70%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 degrees Celsius for three days. On embryonic day six, make a small incision in the chorioallantoic membrane with a 23 gauge needle. Place a seven-day spheroid on the incision using a pipette and return the embryo to the incubator for three days until further experiments.
Place a drop of DPBS to cover the craniotomy. Place the flexible electrode onto the drop of DPBS and gently place the back of the probe with the contact pads onto the mouse's back. Absorb the DPBS drop with a small piece of paper until the probe can lay flat on the dura and follow the curvature of the brain, ensuring the small layer of saline remains below the electrodes to act as a barrier against glue spillover.
Place a small drop of silicone adhesive onto the probe and cover it with a five millimeter 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. Then wait 30 seconds for the silicone to solidify.
To secure the cover glass, quickly apply superglue on its sides and push it down until the glue becomes solid. Apply super glue at the probe's neck using a toothpick taking care that the super glue gets drawn under the neck to provide stable support. Cover the skull with dental cement to build a chronic cap and take special care to cover the edges of the cover glass only.
Lift the back of the probe and apply cement underneath the neck of the probe. Rest of the probe onto the cement before it cures. Push down the neck of the probe gently to place its surface at the same level as the cover glass and not in the way of the microscope objective during the experiment.
Cover the top of the probe neck with not more than 1.5 millimeters of dental cement layer to achieve a firm hold on the probe. Build a cement well presenting a 1.5 millimeter ridge at one to two millimeters around the cover glass to create a basin for the immersion fluid for the two photon imaging. After the cement has cured, administer postsurgical analgesia and keep the animal warm until recovery by wrapping it in a paper towel and placing it close to an infrared light bulb.
The PEDOT:PSS-coated electrodes show the typical capacitive and resistive dominated regions separated by a cutoff frequency, whereas the uncoated electrodes display only capacitive behavior. The growth of the spheroids observed with a brightfield microscope revealed that at least two or three days are needed to obtain spherical and dense spheroids depending on the cell line and the number of cells seeded. In the in ovo model, the graft of spheroids in the chorioallantoic membrane can be assessed by fluorescence microscopy as living cells have intracellular calcium and the vascularization of the tumor can be assessed by injecting a fluorescent dye into the blood vessels.
Compared to the impedance in saline solution, an increase of the impedance is expected in vivo at frequencies above 100 hertz due to the presence of a biological environment. Vascularized neural parenchyma and the tumor infiltration can be observed and characterized through the transparent substrate over weeks by two photon microscopy. The use of transgenic animals expressing fluorescent proteins in cells of interest can demonstrate the minimal inflammatory process induced by electrode implantation alone.
It can show the presence of microglia and monocytes 26 days after implantation pulsed electric fields stimulated electrode. Both peripheral monocyte derived and brain resonant microglial cells were found around and inside the tumor. The critical point for success in the implant procedure is to ensure the sealing and the flatness of the cranial window and to optimize the quality of the contact with probe.
In addition to the intravital imaging, what could consider combining our protocol with other measures and techniques like the samplings and measures of blood work for cytokines, immune status by flow cytometry, or even behavioral analysis. This work paves the way for the use of flexible implantable devices for cancer and opens new prospects for the use of bioelectronic medicine for this chronic disease.
