Dulbecco s phosphate buffer saline pbs

To sterilize the titanium samples, they were autoclaved in water steams at 121 °C and a pressure of 1.5 atmospheres. Then, the samples were placed into the wells of 4-well plates (Nunc, USA). FetMSCs and MG-63 cells were seeded (1 × 10⁵ in 20 µL of DMEM/F12 nutrient medium) on the surface of the samples and maintained for 24 h in CO₂. After incubation, the cells were washed with Dulbecco’s phosphate buffer saline (PBS) (Sigma-Aldrich, Burlington, MA, USA) and fixed in 2.5% glutaraldehyde in phosphate buffer (pH = 7.2, Sigma-Aldrich, Burlington, MA, USA). After 3 days, the samples were washed in a phosphate buffer and successively dehydrated in 30, 50, 70, 90, 96% and absolute ethanol for 30 min each. The final drying was carried out 3 times for 15 min using Leica EM CPD300 at the CO₂ critical point. Finally, the Au coatings with a thickness of about 10 nm were deposited with Leica EM SCD500. The evaluation of the cells’ morphology was performed using a Tescan MIRA3 LMU scanning electron microscope (TESCAN, Brno–Kohoutovice, Czech Republic).

Liposomes were prepared using the ethanol injection method. Lipids were weighed and dissolved in absolute ethanol and subsequently injected into calcium-free Dulbecco’s phosphate buffer saline (PBS; Sigma-Aldrich) preheated to 65 °C reaching a final lipid concentration of 50 mM. The liposomes were extruded five times using a high-pressure Lipex extruder (Northern Lipids, Canada) through 400, 200, and 100 nm polycarbonate-etched membrane (Whatman, Newton, MA, USA) at 65 °C.
For the absorbance measurements, liposomes were diluted 50-fold and measured in a UV-star 96-well microplate (Greiner bio-one, Germany) using the Infinite 200PRO multimode reader (TECAN, Switzerland) controlled with the i-control 1.10 software. Based on the absorbance data, a Lorentzian function was fitted for each of the phospholipids using the curve fitting toolbox in Matlab (version 2021b). The Lorentzian function Eq. (2) is: $L=a/\left({\left(x-b\right)}^{2+c}\right)+d$ Where the constants a, b, c, and d denote the Lorentzian amplitude, center, width, and offset respectively, and x denotes the Lorentzian variable - photon energy.

Electrochemical impedance spectroscopy (EIS) measurements were performed with a Novocontrol Technologies Alpha-A High Performance Frequency Analyzer (Novocontrol Technologies GmbH & Co. KG, Montabaur, Germany). Impedance spectra between 100 Hz and 10 MHz were recorded at 200 distinct frequencies in total, applying a sinusoidal voltage of 10 mV amplitude. For the MEA electrode characterization, EIS was performed in a three-electrode arrangement using one MEA electrode as a working electrode (WE), a platinum wire as a counter electrode (CE), and a commercial Silver/Silver-Chloride (Ag/AgCl) (Dri-Ref short, World Precision Instruments, Inc., Sarasota, FL, USA) electrode as reference electrode (RE). These measurements were carried out in Dulbecco’s phosphate buffer saline (PBS) (purchased from Sigma-Aldrich Chemie GmbH, Munich, Germany).
To identify the optimum AC frequency for the electroporation pulse, we determined the normalized impedance, which describes the contribution of the cell layer to the total impedance. Hereby, two EIS measurements were conducted: one without the cells and one after 24 h, when the cells completely adhered to the MEA. These measurements were performed in cell culture medium and all 60 electrodes of the MEA were short-circuited to reduce the total impedance of the WE.