## 3 Results
### Changes in Electrical Biompedance Induced by the 48 h Secretome but Not the 24 h Secretome  
The bioimpedance spectrum of cell cultures was determined using a 1 MHz focused resonance device, with readings at 200 KHz identified as a useful bandwidth [41]. The treatments with the 24-h and 48-h tumor cell secretomes on MSCs (Figures 8 and 9, respectively) altered the bioimpedance spectra, causing shifts in both the magnitude (\(Z\)-axis) and phase (degree axis), which were represented on logarithmic scales. The use of logarithmic scaling provided a clearer visualization of these modifications, which were more pronounced following treatment with the 48-h secretome, while the 24-h secretome induced only minor changes.  
Within the explored bioimpedance frequency range, changes in the magnitude and phase signatures were indicative of modifications to the volumetric cell structure and cell membrane thickness, respectively. These logarithmic shifts appeared to correlate closely with the morphological and cytoskeletal changes described in Figure 5, as well as with the alterations in actin fiber organization that contributed to microstructural changes upon the membrane surface, as presented in Figure 7.  
Figure 7: Dispositions of the polymerized actin of cytoskeletons in the MSCs cultured with secretomes. Conditions used in the assays with MSCs were as follows: (**a**) DMEM/F12 at 10% FBS, (**b**) 24 h secretome, and (**c**) 48 h secretome. (**d**–**f**) Graphic schematization of how actin fibers contributed to the morphology of the MSCs and their microshapes on the membrane surface. FA: focal adhesion; FC: focal complex; LP: lamelopodium; FP: filopodium; N: nucleus; CT: comet tail; SF: stress fiber; MS: microspike.  
The cell disintegration index (\(Z_{p}\)) based on multifrequency bioimpedance measurements has been widely used to monitor cell structure changes and disruption [42]. Here, we proposed the use of \(Z_{p}\) to quantitively estimate bioimpedance variations with cell morphological changes. \(Z_{p}\) was calculated on the basis of the measurement of the absolute impedance value of the control (\(Z_{c}\)) and secretome-treated (\(Z_{tr}\)) groups (at 24 and 48 h) in the low (1 kHz) and high (150 kHz) frequency ranges, in accordance with [43,44], as follows:  
\[Z_{p}=\frac{\left|Z_{c(1\mathrm{kHz})}-\left|Z_{tr(1\mathrm{kHz})}\right| \right|}{\left|Z_{c(1\mathrm{kHz})}\right|-\left|Z_{tr(150\mathrm{kHz})} \right|} \tag{1}\]  
where the value of \(Z_{p}\) varies between 0 for intact cells and 1 for fully permeabilized cells.  
The results indicate the following index: \(Z_{p(2\mathrm{th})}=0.14\) y \(Z_{p(4\mathrm{8h})}=0.27\). Thus, a greater cell structural change is evident after the 48 h secretome treatment. As a first intuitive approach, secretomes induced early morphological changes related to the migration of MSCs. The modification of the bioimpedance patterns recorded and the \(Z_{p}\) index increments as a function of secretome time suggest that this method can be applied in an innovative way to detect early changes in a cellular population in the clinical diagnostic setting.  
Figure 8: Electrical bioimpedance spectra of magnitude (**a**) and phase (**b**) for cells with the 24 h secretome. \(Z\)—magnitude; \(\theta\)—phase; Hz—frequency. Magnitude and phase signatures display minimal changes with respect to the control conditions and show \(Z_{p(2\mathrm{th})}=0.14\).  
Figure 9: Electrical bioimpedance spectra of magnitude (**a**) and phase (**b**) for cells with the 48 h secretome. \(Z\)—magnitude; \(\theta\)—phase; Hz—frequency. Magnitude and phase signatures display greater changes with respect to the control conditions and show \(Z_{p(4\mathrm{8h})}=0.27\).