*2.1. Theory*

Bioimpedance, as a passive electrical property, is described as the capability of biological tissue to impede electric current. Bioimpedance measurements detect the response to electrical activity (potential or current). Bioimpedance is a complex quantity, mainly determined by the resistance (R) of the total amount of body water and by the capacitance of the cell membrane [40]. Electrical Impedance (Z) is defined by the ratio of the voltage (V) to the current (I), and is quite similar to resistance. The basic difference is that impedance extends to the frequency domain, and thus, is used in AC circuits, while resistance mainly refers to DC applications. The equation for the calculation of electrical impedance is:

$$\mathbf{Z} = \mathbf{V}/\mathbf{I} \tag{1}$$

The determination of electrical impedance requires not only the application of an alternating current across a biological tissue, but also the measurement of the consequential differential voltage of the tissue sample, described in the following equations:

$$\mathbf{I}(\omega) = \mathbf{I}\_0 \cdot \cos(\omega \mathbf{t} + \theta) \tag{2}$$

$$\mathbf{V}(\omega) = \mathbf{V}\_0 \cdot \sin(\omega \mathbf{t} + \psi) \tag{3}$$

where I0 and V0 represent the amplitude θ and ψ the phase of the current and voltage signal, respectively. Taking into account that both I0 and V0 are calculated at the same angular frequency, i.e., ω = 2πf, electrical impedance can be described as Z(ω) = (V(ω))/(I(ω)) [41]. The expression of Z (Equation (1)) as a complex function can be used either as the modulus of the absolute value and the phase shift, or as the real part, R, representing resistance, and the imaginary part, X, representing capacitance and inductance, respectively [42]. In the case of direct current (DC) application, the imaginary part would be zero. The inverse of the impedance is called admittance (Y) and describes the current flow. Impedance and admittance constitute AC parameters, and both are frequency dependent. The EIS measurement procedure involves the characterization of the complex impedance over a wide range of frequencies, as shown in Equation (3):

$$|\mathbf{Z}(\omega) = |\mathbf{Z}|(\cos \varphi + \mathbf{j} \sin \varphi) = \mathbf{R} + \mathbf{j} \,\, \mathbf{X} \tag{4}$$

## *2.2. Cell Culture*

SK-N-SH neuroblastoma cells (ATCC® HTB-11™) were cultured under standard conditions (37 ◦C, 5% CO2) in 90% Minimum Essential Medium (MEM) (Eagle) with Earle's balanced salt solution (BSS) (Biowest, Nuaillé, France) and fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) to a final concentration of 10%, 2 mM l-glutamine, 1.5 g/<sup>L</sup> sodium bicarbonate, 0.1 mM non-essential amino acids, 1 U μg<sup>−</sup><sup>1</sup> antibiotics (penicillin/streptomycin), and 1.0 mM sodium pyruvate (Biowest, Nuaillé, France). HEK293 (ATCC® CRL-1573™), HeLa (ATCC® CRM-CCL-2™) and MCF-7 (ATCC® HTB-22™) cell lines were grown in Dulbecco's Modified Eagle Medium (Biochrom Gmbh, Berlin, Germany), supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA, USA), 2 mM l-glutamine, 0.5 mM sodium pyruvate, and 1% Penicillin-Streptomycin (Biowest, Nuaillé, France) in T-75 flasks (Sarstedt AG & Co. KG, Nümbrecht, Germany). Subcultivation was done in a 1:10 ratio. Cells were detached from culture flasks by treatment with trypsin-EDTA for 3–10 min. After detachment, they were resuspended in the culture medium to inactivate any remaining trypsin activity. After centrifugation for 5 min (1000 rpm), they were resuspended in the medium at concentrations of 106, 2 × 106, and 4 × 10<sup>6</sup> cells/mL.

## *2.3. Cells Preparation*/*Immobilization*

Cell immobilization was performed in calcium alginate as an immobilization matrix. Briefly, sodium alginate in 1.5% concentration, sterilized by autoclave (121 ◦C, 20 min), was mixed with 5 × 104, 105, and 2 × 10<sup>5</sup> cells to a 0.75% final concentration and poured together in the well. Then, a 1% CaCl2 gelling solution was added for 10 s for cross-linking, and washed with phosphate buffered saline (PBS). After washing, the calcium alginate scaffolds with the cells were incubated in the culture medium. The next day, the culture medium was removed and replaced with 1% FBS medium and 1% FBS with different concentrations 5-FU (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany).

#### *2.4. Cell Viability Assay*

Cell viability was evaluated by a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [43] with 5-FU as the positive control. The concentration of 5-FU for each cell line, indicated in Table 1, was selected based on previously published data [44–48]. These concentrations gave at least 30% inhibition in cell proliferation after 24 h incubation. The next day, the cells were treated with 0.5 mg/mL MTT (Duchefa Biochemie, Haarlem, The Netherlands) and incubated with dye for 3 h. After incubation, the medium was removed and the cell containing alginate scaffolds were solubilized with 0.1 M ethylenediaminetetraacetic acid per well. Cell morphology observations were performed with an inverted microscope (ZEISS Axio Vert.A1, Carl Zeiss Microscopy, LLC, White Plains, NY, USA), and pictures were processed using the ZEN lite software. The optical absorbance was measured with a PowerWave240 plate reader (BioTek, Winooski, VT, USA) at 560 nm. The experiment was repeated independently three times for each treatment, and the mean results were expressed as the average OD for each treatment. All values were presented as means ± SD, and significance testing in the comparisons was based on Student's T-tests for pairs, as they did not follow a normal distribution. The Student's T-test gives the probability that the difference between the two means is caused by chance. *p* values < 0.05 were considered to be statistically significant.


**Table 1.** 5-FU concentrations added to each cell culture.

## *2.5. Experimental Setup*

In order to perform the bioelectrical measurements, a specifically designed electrode-based system was fabricated. Figure 1a illustrates the experimental setup of the system used for the measurement procedure. More specifically, two gold-coated (Au) electrodes were placed vertically into a custom-made, transparent, 3D-printed PETG well that was designed to be used as a cell cultivation vessel. The application of 3D printing technology to the assembly of culture systems can provide an appropriate environment for cell growth which is able to mimic physiological and realistic cell phenotypes [49]. The well model was designed using the 123D Design software (Autodesk, San Rafael, CA, USA). A Cel Robox 3D printer device was utilized for the printing procedure, applying the fused deposition modeling technique (FDM). By this method, the filament that passes through the heated print head is laid on a construction platform in a layer-by-layer fashion until the object's form is complete [50]. The nozzle diameter of the print head was 0.4 mm and the printing temperature for PETG was 190 ◦C [38]. The PETG filament was obtained by the Formfutura BV (Nijmegen, The Netherlands); the filament diameter was 1.75 ± 0.05 mm. After the printing process, the wells were sterilized with 70% (v/v) ethanol for 10 min, and then dried for 2 h under a sterile hood. The electrodes were connected to the handheld LCR meter U1733C (Figure 1b) from Keysight Technologies (Santa Rosa, CA, USA); the instrument is able to measure at three frequencies (1 KHz, 10 KHz, and 100 KHz) for the direct extraction of impedance magnitude of the sample tested. For impedance measurements, a voltage of 0.74 Vrms ± 50 mVrms was applied via the two terminals to the gold-coated electrodes. The best sampling rate of the instrument was 1 Hz (one measurement per sec); each measurement lasted one minute; thus, the total values obtained for each run were 60, with a measurement frequency of 1 Hz. All data were normalized values, presented as the mean of the absolute (ABS) value of the control (plain cell culture medium) minus the absolute value of cells, for both cases (treated or untreated with 5-FU ± SD), as shown in Equation (5). Significance testing in comparisons was based on Student's T-tests for pairs, and *p* values < 0.05 were considered to be statistically significant.

#### Normalized value = mean (|control-cell value|) (5)

**Figure 1.** Experimental setup. (**a**) Representation of the cell chamber filled with 3D cell immobilization matrix; (**b**) Connection of the LCR meter to the 3D printed well.
