# Selector+
## 3 Results and Discussion  
### Characterization of Expanded Cell Line  
The cancer cell lines HL60 and SW620 were analyzed using Celector(r) to monitor their physical composition and the possible modification of their homogeneity. These two cell lines were chosen to observe possible differences in culture expansion because of their adhesion properties: SW620 are adherent cells, small in dimension with a star shape, and they grow in very tight connection with each other and with a slower proliferative rate; HL60 cells grow in suspension with a higher proliferative rate compared to SW620, and differences in morphology and dimension can be observed during passages in culture. The first run served to obtain the sample fractogram, the fingerprint of the sample, and to decide the time interval of sorting. Successively, further runs were performed to obtain results in triplicate, and one fraction was consecutively collected to obtain the right number of cells. The time interval for HL60 was set between the 4 th and 10 th minutes of analysis, depleting the living cells from the unwanted material such as cell debris and dead cells (Figure 1A). The curve representing the number of cells as a function of time showed a similar-normal distribution, with the peak in the middle of the curve: 60% of cells eluting in the ascending portion and 40% in the descending portion of the curve. The collected cells (POST) were centrifuged and expanded in vitro following the same culture condition of the control (CTRL), the entire cell population. At every passage in culture, after enzymatic detachment, CTRL- and POST-derived cells were counted and analyzed at the instrument to monitor the fractogram, and successively, their physical characteristics. One passage after the sorting, CTRL and POST cells did not show differences in their fractograms (Figure 1B), but the ratio between the initial eluate compared to the final (F1 vs. F2) (Figure 1D) was reversed: 40% to F1, and 60% of cells were now in the second half, namely F2. This trend increased further after a couple of passages. At p5, 20% of cells were in F1 for the CTRL and 15% for the POST cells (Figure 1D), and the majority in F2. The total time of elution between p2 and p5 persisted. However, at p5, there was a clear shift in the cells' presence towards the fraction F2 (Figure 1C). This shift denoted a change in the process of in vitro cell expansion. Due to the fact that FFF is based on physical characteristics, we investigated the dimensions of cells that can be obtained by post-processing image analysis. The average of all eluted cells showed a diameter of 25 um at p1, 26 um and 27 um at p2 for CTRL- and POST-cells, respectively, and an even higher enlargement at p5, with the CTRL reaching 30 um and POST 28 um (Figure 1E). Splitting the population into the two hypotheticals fractions, cells were slightly larger in F2 at p2, and this difference was intensified at p5 (Figure 1F). The curve representing the average of the diameter, in Figure 1G, represents the cell culture behavior observed in the cell fractogram,showing a similar-normal distribution for cells at p1 and a p2, while the majority of cells in F2 exhibited a higher diameter at p5. Generally, based on FFF principles, larger cells exit at the beginning of the analysis, followed by smaller cells. A HL60 dimensional analysis showed a higher dimension of F2 cells compared to F1. Therefore, the density of the cells could be the parameter to distinguish F1 cells from F2 cells. A deeper investigation of the intracellular composition should be performed to understand the state of the cells, such as cell-cycle phase, mitochondrial activity, and protein content [22; 23]. A different behavior was observed when adherent SW620 cells were analyzed during their in vitro expansion. SW620 cells showed a similar-normal distribution as a rather homogenous population (Figure 2A) with an opposite trend compared to HL60 cells: 40% of cells eluted in the first part of the curve, F1, and 60% in the descendant slope, F2 (Figure 2D).