**1. Introduction**

Cancer is the main cause of death in many countries, as it appears in different types, most commonly affecting women (e.g., cervical, breast, and lung adenocarcinoma cancers) [1,2]. In medical treatment, doctors take steps to anticipate the development of the disease (primary prophylaxis) or to minimize its further development (secondary prophylaxis) [3]. Considering secondary prophylaxis measures, sophisticated processes are required to detect possible cellular disorders at the very earliest stages of the disease's incubation period, taking into account the dependence of the timeliness with which the disease is detected [4]. Due to the fact that many cancer diagnostic methods combined with radiological, surgical biopsy, and pathological assessments of tissue samples based on immunohistochemical and morphological characteristics [5] are time-consuming, invasive, and complicated, and require rigorous laboratory conditions, new cancer detection methods are being developed which are minimally invasive, more reliable, cheaper, and easier to use [6].

Chemotherapy, newer immunotherapy, and targeted therapy constitute the various treatment strategies that have been proposed and modified in order to increase effectiveness and precision [7,8]. Although many approaches for cancer treatments have been developed successfully, sooner or later, resistance among the subgroups of cancer cells will emerge as a hurdle to the e fficacy of most current therapeutic approaches [9]. One of the most commonly used drugs for cancer treatment is 5-fluorouracil (5-FU). This compound is used in the treatment of many types of cancer, including breast cancer, colon cancer, skin cancer, etc., as it intercalates in nucleoside metabolism, leading to cytotoxicity and cell death [10].

Electrical impedance spectroscopy (EIS) is a technique that measures the electrical impedance of living cells in order to identify various cell types. This technique can be used for the successful separation of pathological cells from normal ones, taking into account the electrophysiological properties of cells based on the frequency range [11,12]. Cell electrical impedance can identify the physical, mechanical, and biochemical functions of living biological cells. EIS focuses on the analysis and discrimination of cancer cells, utilizing the fact that impedance measurements represent an e ffective approach for cell characterization based on the electrical responses over a particular frequency domain. As the impedance value of several tissue parameters (e.g., morphology, growth, and di fferentiation) vary with the frequency of the applied signal, an impedance analysis conducted over a wide frequency range provides more information about the tissue interiors, which helps us to better understand the biological tissues physiology, anatomy, and pathology [13]. For example, researchers have used the impedance technique to measure three dimensional cell cultures for four breast cell lines [14–16].

Recently, bioimpedance has been able to provide in-depth biological measurement analyses from the cell-level to DNA [17,18]. The evaluation of parameters such as cell adhesion, di fferentiation, spreading, morphology, growth, motility, and death for any adherent cell type is possible by monitoring the impedance changes at the contact point between the cells and electrodes [19]. In addition, bioimpedance research can nominate the pathological status of a single cell, and also be used to determine the occurrence of bacterial infections, toxicity, and changes of environmental parameters, and in the direct or indirect detection of compounds and other factors [20]. Critical changes in cellular behavior, such as the integrity of the extracellular membrane, morphology, as well as alterations in intracellular structure, significantly influence the corresponding impedance level which can be detected quickly and cost-e ffectively using electrodes [13,19]. Thus, impedance measurements can also be used in studying cell viability, which provides an alternative to slow and invasive traditional cytotoxicity assays [21].

Biomaterial research for drug development, cell culture, and tissue regeneration applications aims to mimic the natural extracellular matrix (ECM) in order to bridge the gap between in vivo and in vitro environments [22]. In the body, nearly all tissue cells are supported by an ECM that comprises a complex, three-dimensional (3D), fibrous mesh network of collagen and elastic fibers integrated into a high hydrated, gel-like material containing proteoglycans, glycosaminoglycans, and glycoproteins. This complex system is responsible for the triggering of various biochemical and physical signals [23]. In practice, most cell culture studies are carried out using cells cultured as two-dimensional (2D) monolayers on hard plastic surfaces due to the convenience, ease, and high cell proliferation that these culture techniques provide. On the other hand, cell adaption to an artificial monolayer culture on an inflexible surface would lead to metabolic and functional alterations, resulting in behavior di fferent from the in vivo environment [23]. Thus, research is focused on developing more controllable 3D cell culture matrices resembling, as much as possible, the in vivo conditions that are able to support cell growth, di fferentiation, and organization. Bearing in mind all the above, we can define 3D cell culture as the integration of cells into a hydrogel matrix in order to receive signals from the sca ffold and surrounding cells [23,24]. This procedure initially necessitates a cellular suspension in a hydrogel precursor solution, and then entrapment through a gel initiation reaction that leads to the formation of covalently- or noncovalently-linked molecules [25,26].

A grea<sup>t</sup> number of synthetic and natural polymers can be used for cell entrapment gelled into hydrophilic matrices under mild conditions with minimal loss of viability [27]. The properties of the gel, i.e., either hydrophobic either hydrophilic, and its porosity, can be regulated. The entrapment of cells constitutes one of the most widely used methods for living cell immobilization within spherical

beads of calcium alginate. This method is considered successful due to the fact that immobilization is a simple, quick and cost-effective technique, and is usually performed under very mild conditions [28].

Three-dimensional (3D) printing is a cheap additive layer manufacturing technology that is able to create sophisticated and complex-shaped bodies in a short time, especially for rapid prototyping engineering applications [29–31]. In many cases, 3D printing technology uses polymers, depending on the specific characteristics that are required for the microfabricated devices (e.g., polycarbonate (PC), PLA, nylon, polymethyl methacrylate (PMMA), polystyrene, and polyethylene terephthalate glycol (PETG)) [32–35]. Depending on the area of interest, 3D printing technology is increasing rapidly and is extensively used in many fields, such as bio-printing, medical devices, the automotive industry, soft sensors and actuators, space, art and jewelry, education, and tissue printing [36,37]. PETG constitutes a copolymer known for its biocompatibility, chemical resistance, recyclability, and transparency [38]. It can be used in different applications in the food and medical industry, with an acceptable flammability rating [39]. Unfortunately, it presents low resistance to ultraviolet (UV) light and performs weakly against frictional contact and scratching.

The aim of this study is to develop a proof-of-concept bioelectrical profiling assay to study the reaction of various cancer cell lines exposed to a common anticancer drug as a function of the cell population density. For comparison purposes, bioelectrical impedance-based measurements were taken on both untreated immobilized cells and on cells treated with 5-FU. Thus, two gold-plated (Au) electrodes were embedded in a 3D-printed PETG well for impedance measurements on four cancer cell lines (SK-N-SH, HEK293, HeLa and MCF-7) immobilized in calcium alginate matrix. Cell cultures were realized in three population densities tested with various frequencies. In this way, a more detailed application of the bioelectrical analysis on an in vitro system for monitoring different responses between various cancer cells (control and treated with 5-FU) was possible.

#### **2. Materials and Methods**
