**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.

**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.

**Figure 2.**
Panoramic view of SK-N-SH immobilized cells in 3D matrix after treatment with MTT for 24 h, showing the viability in three different population densities: (**a**) 50,000 cells; (**b**) 100,000 cells; and (**c**) 200,000 cells. Scale bars = 50 μm.

**Figure 2.**
Panoramic view of SK-N-SH immobilized cells in 3D matrix after treatment with MTT for 24 h, showing the viability in three different population densities: (**a**) 50,000 cells; (**b**) 100,000 cells; and (**c**) 200,000 cells. Scale bars = 50 μm.

**Figure 3.**
Panoramic view of HEK293 immobilized cells in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities: (**a**) 50,000 cells; (**b**) 100,000 cells; and (**c**) 200,000 cells. Scale bars = 50 μm.

**Figure 3.**
Panoramic view of HEK293 immobilized cells in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities: (**a**) 50,000 cells; (**b**) 100,000 cells; and (**c**) 200,000 cells. Scale bars = 50 μm.

**Figure 4.**
Panoramic view of HeLa immobilized cells in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities: (**a**) 50,000 cells; (**b**) 100,000 cells; and (**c**) 200,000 cells/100 μL. Scale bars = 50 μm.

**Figure 4.**
Panoramic view of HeLa immobilized cells in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities: (**a**) 50,000 cells; (**b**) 100,000 cells; and (**c**) 200,000 cells/100 μL. Scale bars = 50 μm.

**Figure 5.**
Panoramic view of MCF-7 immobilized cells in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities: (**a**) 50,000 cells; (**b**) 100,000 cells; and (**c**) 200,000 cells/100 μL. Scale bars = 50 μm.

**Figure 5.**
Panoramic view of MCF-7 immobilized cells in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities: (**a**) 50,000 cells; (**b**) 100,000 cells; and (**c**) 200,000 cells/100 μL. Scale bars = 50 μm.

**Figure 6.**
Cellular viability of SK-N-SH cells immobilized in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities (50,000, 100,000, and 200,000 cells/100 μL) ± STD: (**a**) untreated cells (control); (**b**) cells treated with 5-FU. ## < 0.01 significantly different from 100,000 cells/100 μL.

**Figure 6.**
Cellular viability of SK-N-SH cells immobilized in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities (50,000, 100,000, and 200,000 cells/100 μL) ± STD: (**a**) untreated cells (control); (**b**) cells treated with 5-FU. ## < 0.01 significantly different from 100,000 cells/100 μL.

**Figure 7.**
Cellular viability of HEK293 cells immobilized in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities (50,000, 100,000, and 200,000 cells/100 μL) ± STD: (**a**) untreated cells (control); (**b**) cells treated with 5-FU. * < 0.05 significantly different from 50,000 cells, ## < 0.01 significantly different from 100,000 cells/100 μL.

**Figure 7.**
Cellular viability of HEK293 cells immobilized in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities (50,000, 100,000, and 200,000 cells/100 μL) ± STD: (**a**) untreated cells (control); (**b**) cells treated with 5-FU. * < 0.05 significantly different from 50,000 cells, ## < 0.01 significantly different from 100,000 cells/100 μL.

**Figure 8.**
Cellular viability of HeLa cells immobilized in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities (50,000, 100,000, and 200,000 cells/100 μL) ± STD: (**a**) untreated cells (control); (**b**) cells treated with 5-FU. * < 0.05, ** < 0.01 significantly different from 50,000 cells, # < 0.05, ### < 0.001 significantly different from 100,000 cells/100 μL.

**Figure 8.**
Cellular viability of HeLa cells immobilized in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities (50,000, 100,000, and 200,000 cells/100 μL) ± STD: (**a**) untreated cells (control); (**b**) cells treated with 5-FU. * < 0.05, ** < 0.01 significantly different from 50,000 cells, # < 0.05, ### < 0.001 significantly different from 100,000 cells/100 μL.

**Figure 9.**
Cellular viability of MCF-7 cells immobilized in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities (50,000, 100,000, and 200,000 cells/100 μL) ± STD: (**a**) untreated cells (control); (**b**) cells treated with 5-FU. * < 0.05, ** < 0.01, *** < 0.001 significantly different from 50,000 cells.

**Figure 9.**
Cellular viability of MCF-7 cells immobilized in 3D matrix after treatment with MTT for 24 h showing the viability in three different population densities (50,000, 100,000, and 200,000 cells/100 μL) ± STD: (**a**) untreated cells (control); (**b**) cells treated with 5-FU. * < 0.05, ** < 0.01, *** < 0.001 significantly different from 50,000 cells.

**Figure 10.**
Normalized values of the mean impedance magnitude for untreated (control) immobilized SK-N-SH cancer cell lines tested at three frequencies (1 KHz, 10 KHz, 100 KHz) for three different population densities ± STD: (**a**) 50,000 cells; (**b**) 100,000 cells and (**c**) 200,000 cells/100 μL.

**Figure 10.**
Normalized values of the mean impedance magnitude for untreated (control) immobilized SK-N-SH cancer cell lines tested at three frequencies (1 KHz, 10 KHz, 100 KHz) for three different population densities ± STD: (**a**) 50,000 cells; (**b**) 100,000 cells and (**c**) 200,000 cells/100 μL.

**Figure 11.**
Normalized values of the mean impedance magnitude for untreated (control) immobilized HEK293 cancer cell lines tested at three frequencies (1 KHz, 10 KHz, 100 KHz) for three different population densities ± STD: (**a**) 50,000 cells; (**b**) 100,000 cells and (**c**) 200,000 cells/100 μL.

**Figure 11.**
Normalized values of the mean impedance magnitude for untreated (control) immobilized HEK293 cancer cell lines tested at three frequencies (1 KHz, 10 KHz, 100 KHz) for three different population densities ± STD: (**a**) 50,000 cells; (**b**) 100,000 cells and (**c**) 200,000 cells/100 μL.

**Figure 12.**
Normalized values of the mean impedance magnitude for untreated (control) immobilized HeLa cancer cell lines tested at three frequencies (1 KHz, 10 KHz, 100 KHz) for three different population densities ± STD: (**a**) 50,000 cells; (**b**) 100,000 cells and (**c**) 200,000 cells/100 μL.

**Figure 12.**
Normalized values of the mean impedance magnitude for untreated (control) immobilized HeLa cancer cell lines tested at three frequencies (1 KHz, 10 KHz, 100 KHz) for three different population densities ± STD: (**a**) 50,000 cells; (**b**) 100,000 cells and (**c**) 200,000 cells/100 μL.

**Figure 13.**
Normalized values of the mean impedance magnitude for untreated (control) immobilized MCF-7 cancer cell lines tested at three frequencies (1 KHz, 10 KHz, 100 KHz) for three different population densities: (**a**) 50,000 cells; (**b**) 100,000 cells and (**c**) 200,000 cells/100 μL.

**Figure 13.**
Normalized values of the mean impedance magnitude for untreated (control) immobilized MCF-7 cancer cell lines tested at three frequencies (1 KHz, 10 KHz, 100 KHz) for three different population densities: (**a**) 50,000 cells; (**b**) 100,000 cells and (**c**) 200,000 cells/100 μL.

**Figure 14.**
Normalized values of the mean impedance magnitude for control immobilized SK-N-SH cells (blue bars) and immobilized SK-N-SH cells treated with 5-FU (yellow bars), tested at three different cell population densities ± STD: (**a**) 50,000; (**b**) 100,000 and (**c**) 200,000/100 μL for three different frequencies (1 KHz, 10 KHz, and 100 KHz).

**Figure 14.**
Normalized values of the mean impedance magnitude for control immobilized SK-N-SH cells (blue bars) and immobilized SK-N-SH cells treated with 5-FU (yellow bars), tested at three different cell population densities ± STD: (**a**) 50,000; (**b**) 100,000 and (**c**) 200,000/100 μL for three different frequencies (1 KHz, 10 KHz, and 100 KHz).

**Figure 15.**
Normalized values of the mean impedance magnitude for control immobilized HEK293 cells (blue bars) and immobilized HEK293 cells treated with 5-FU (yellow bars), tested at three different cell population densities ± STD: (**a**) 50,000; (**b**) 100,000 and (**c**) 200,000/100 μL for three different frequencies (1 KHz, 10 KHz, and 100 KHz).

**Figure 15.**
Normalized values of the mean impedance magnitude for control immobilized HEK293 cells (blue bars) and immobilized HEK293 cells treated with 5-FU (yellow bars), tested at three different cell population densities ± STD: (**a**) 50,000; (**b**) 100,000 and (**c**) 200,000/100 μL for three different frequencies (1 KHz, 10 KHz, and 100 KHz).

**Figure 16.**
Normalized values of the mean impedance magnitude for control immobilized HeLa cells (blue bars) and immobilized HeLa cells treated with 5-FU (yellow bars), tested at three different cell population densities ± STD: (**a**) 50,000; (**b**) 100,000 and (**c**) 200,000/100 μL for three different frequencies (1 KHz, 10 KHz, and 100 KHz).

**Figure 16.**
Normalized values of the mean impedance magnitude for control immobilized HeLa cells (blue bars) and immobilized HeLa cells treated with 5-FU (yellow bars), tested at three different cell population densities ± STD: (**a**) 50,000; (**b**) 100,000 and (**c**) 200,000/100 μL for three different frequencies (1 KHz, 10 KHz, and 100 KHz).

**Figure 17.**
Normalized values of the mean impedance magnitude for control immobilized MCF-7 cells (blue bars) and immobilized MCF-7 cells treated with 5-FU (yellow bars), tested at three different cell population densities ± STD: (**a**) 50,000; (**b**) 100,000 and (**c**) 200,000/100 μL for three different frequencies (1 KHz, 10 KHz, and 100 KHz).

**Figure 17.**
Normalized values of the mean impedance magnitude for control immobilized MCF-7 cells (blue bars) and immobilized MCF-7 cells treated with 5-FU (yellow bars), tested at three different cell population densities ± STD: (**a**) 50,000; (**b**) 100,000 and (**c**) 200,000/100 μL for three different frequencies (1 KHz, 10 KHz, and 100 KHz).

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

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

| SK-N-SH | HEK293 | HeLa | MCF-7 |
---|

5-FU concentration (μΜ) | 7.5 | 20 | 150 | 150 |

**Table 2.**
Significant differences (Student’s T-test) between cell populations before and after treatment with 5-FU. **< 0.01, *** < 0.001.

**Table 2.**
Significant differences (Student’s T-test) between cell populations before and after treatment with 5-FU. **< 0.01, *** < 0.001.

| 50,000 Cells | 100,000 Cells | 200,000 Cells |
---|

SK-N-SH | - | ** | - |

HEK293 | - | ** | - |

HeLa | - | *** | - |

MCF-7 | - | - | - |

**Table 3.**
Significant differences (Student’s T-test) in cell viability between different cell lines X cell population densities before and after treatment with 5-FU. * < 0.05, **< 0.01, *** < 0.001.

**Table 3.**
Significant differences (Student’s T-test) in cell viability between different cell lines X cell population densities before and after treatment with 5-FU. * < 0.05, **< 0.01, *** < 0.001.

| Cells | Cells Treated with 5-FU |
---|

| 50,000 Cells | 100,000 Cells | 200,000 Cells | 50,000 Cells | 100,000 Cells | 200,000 Cells |
---|

SK-N-SH–HEK293 | - | - | * | - | - | * |

SK-N-SH–HeLa | - | - | ** | - | - | - |

SK-N-SH–MCF-7 | - | * | *** | - | *** | * |

HEK293–HeLa | - | * | - | - | ** | * |

HEK293–MCF-7 | - | * | - | - | ** | - |

HeLa–MCF-7 | - | ** | * | - | ** | * |

**Table 4.**
Significant differences (Student’s T-test) between population densities for the SK-N-SH cell line before and after treatment with 5-FU. **< 0.01, *** < 0.001.

**Table 4.**
Significant differences (Student’s T-test) between population densities for the SK-N-SH cell line before and after treatment with 5-FU. **< 0.01, *** < 0.001.

| Cells | Cells Treated with 5-FU |
---|

| 1 KHz | 10 KHz | 100 KHz | 1 KHz | 10 KHz | 100 KHz |
---|

50,000–100,000 | *** | *** | ** | ** | *** | *** |

50,000–200,000 | - | *** | - | *** | *** | *** |

100,000–200,000 | *** | *** | - | *** | ** | *** |

**Table 5.**
Significant differences (Student’s T-test) between population densities for HEK293 cell line before and after treatment with 5-FU. * < 0.05, **< 0.01, *** < 0.001.

**Table 5.**
Significant differences (Student’s T-test) between population densities for HEK293 cell line before and after treatment with 5-FU. * < 0.05, **< 0.01, *** < 0.001.

| Cells | Cells Treated with 5-FU |
---|

| 1 KHz | 10 KHz | 100 KHz | 1 KHz | 10 KHz | 100 KHz |
---|

50,000–100,000 | *** | *** | ** | *** | *** | ** |

50,000–200,000 | *** | *** | * | *** | - | ** |

100,000–200,000 | *** | *** | *** | *** | * | ** |

**Table 6.**
Significant differences (Student’s T-test) between population densities for HeLa cell line before and after treatment with 5-FU. * < 0.05, **< 0.01, *** < 0.001.

**Table 6.**
Significant differences (Student’s T-test) between population densities for HeLa cell line before and after treatment with 5-FU. * < 0.05, **< 0.01, *** < 0.001.

| Cells | Cells Treated with 5-FU |
---|

| 1 KHz | 10 KHz | 100 KHz | 1 KHz | 10 KHz | 100 KHz |
---|

50,000–100,000 | *** | *** | * | *** | *** | ** |

50,000–200,000 | *** | ** | *** | - | *** | *** |

100,000–200,000 | * | *** | *** | *** | *** | *** |

**Table 7.**
Significant differences (Student’s T-test) between population densities for MCF-7 cell line before and after treatment with 5-FU. * < 0.05, **< 0.01, *** < 0.001.

**Table 7.**
Significant differences (Student’s T-test) between population densities for MCF-7 cell line before and after treatment with 5-FU. * < 0.05, **< 0.01, *** < 0.001.

| Cells | Cells Treated with 5-FU |
---|

| 1 KHz | 10 KHz | 100 KHz | 1 KHz | 10 KHz | 100 KHz |
---|

50,000–100,000 | - | *** | ** | *** | *** | *** |

50,000–200,000 | ** | *** | *** | *** | *** | *** |

100,000–200,000 | * | *** | * | *** | *** | ** |