1. INTRODUCTION
The traditional two-dimensional (2D) cell culture has been extensively utilized over the past two decades, greatly enhancing our comprehension of disease mechanisms and preclinical evaluation of pharmacological compounds [1–5]. However, this setting may deviate appreciably from the in vivo response in different disease models due to the mismatch in cell morphology, polarity, homogeneity, and heterogeneous tissue interactions [6–10].
These limitations are often more pronounced in cancer cells due to their complex environment, which involves interactions among adjacent cancer cells, stromal cells, and the extracellular matrix scaffold, collectively known as the tumor microenvironment [10–13]. This microenvironment gives rise to unique cascading signaling pathways, in addition to the spatial variation in cell responses to treatment between the inner core and the periphery, leading to variable access to blood supply and reducing treatment effectiveness [13–17]. This disparity in treatment response may significantly impact the results of preclinical screening, where cancer cells grown in spheres were found to be more resistant to cytotoxic drugs than cells in dispersed culture or monolayer [9,18,19]. Consequently, a higher concentration of treatment is required to arrest the growth rate of cells compared to the dose needed in the tissue culture model. Therefore, developing a controllable and more realistic culture system that mimics the in vivo bioactivities of cells is necessary, given the limitations of the 2D setting. This study developed a three-dimensional (3D) cell culture system that aims to reproduce certain structural aspects of the tumor microenvironment [20–22]. Utilizing these alternatives may facilitate a deeper understanding of cell metabolism and the interactions between biological molecules and tissues in diseases such as cancer [23]. One of the changes in the 3D setting is the alteration of cancer cell morphology due to cytoskeletal rearrangements, in which cells adopt an asymmetric shape with different arrangements of surface receptors and cellular interactions, leading to enhanced proliferation of cancer cells [24]. Hepatocytes are characterized by their heterogeneous extracellular matrix, which is difficult to replicate using simple 2D cell culture techniques [25–27]. The use of spheroid HepG2 cells also appeared to be more differentiated than HepG2 cells cultured in a monolayer system, possessing the mature metabolic function of liver cells. Hydrogel microparticles have been used to encapsulate cancer cells; for example, alginate particles allow for precise control over the initial cell number, stiffness, and bead size, thereby better mimicking the in vivo progression of cancer cells [28].
Alginate is an anionic natural polysaccharide polymer generated from brown algae cell walls. It is composed of 1,4-linked β-D-mannuronic acid and 1,4 α-L-guluronic acid. Alginate is commonly used as a coating material in drug delivery systems due to its crosslinking characteristics with metal ions, such as Ca2+, and it also exhibits biocompatible properties [29]. It is commonly used as a blending system to enhance the transport of Poly(lactic-co-glycolic acid) nanoparticles across mucosal barriers. The alginate systems have also contributed to tissue engineering preparations with the capacity to regenerate or replace biological tissues [30]. As one of the most rapidly growing approaches in the life sciences, 3D cell culture systems based on alginate beads have potential applications in research and the healthcare industry, providing new matrices and scaffolds [14].
Natural compounds have emerged as promising anticancer agents because of their low toxicity and therapeutic properties. Thymoquinone (TQ) is the predominant active component of black cumin seed oil (Nigella sativa), with potent antioxidant properties and well-demonstrated cytotoxic properties in cancer cell culture systems and animal models [31]. The cytotoxic effects of TQ are attributed to its ability to modulate apoptosis in cancer cells. This procedure entails increasing the Bax/BCL-2 ratio and eliciting cell cycle arrest in particular cancer cell types [32,33].
The purpose of this work was to develop a 3D alginate bead-based HepG2 cell culture using electrospray and to investigate the effect of 3D cell culture systems on HepG2 cell viability and the cytotoxicity of TQ in the cells compared to 2D monolayer culture.
2. MATERIALS AND METHODS
2.1. Materials
In this investigation, TQ (Sigma Aldrich, USA) was employed. We purchased calcium alginate (IL-6G, nominal viscosity = 300 mPa.s, molecular weight = 4.8 × 106 ± 1.8 × 105 Da) from Kimica Algin in Tokyo, Japan. We purchased phosphate-buffered saline (PBS) from R&M in China. Sigma Aldrich, USA, provided Trypsin-EDTA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Trypan blue solution, fetal bovine serum (FBS), and high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM). The HepG2 hepatocellular cancer cell line was obtained from the American Type Culture Collection.
2.2. Cell culture maintenance
HepG2 liver cancer cells were cultivated in DMEM high-glucose medium supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were grown at 37°C in an atmosphere with 5% CO2. All tests were conducted using HepG2 cells between passages 5 and 15. Before being subcultured or seeded into microplates, the cells were rinsed three times with 2 ml of PBS. The cells were then detached using 1 ml of trypsin and incubated for 5 minutes at 37°C with 5% CO2. Five milliliters of complete growth medium (DMEM with 10% FBS) were added to neutralize trypsin. A hemocytometer was used to measure the cell viability and density following trypsinization using the Trypan blue assay. Since the HepG2 cell line employed in this investigation is a commercially accessible human hepatocellular carcinoma cell line, ethical approval was not necessary.
2.3. 2D cytotoxicity assay
The MTT assay was used to evaluate the cytotoxicity of TQ on HepG2 cells. HepG2 cells were seeded into a 96-well plate at a density of 1 × 104 cells per well in 200 µl of complete culture medium and incubated at 37°C with 5% CO2 for 24 hours. After incubation, the culture medium was aspirated and replaced with fresh medium containing TQ at concentrations ranging from 15.62 to 2,000 µg/ml. This extended concentration range was necessary to determine the IC50 value in the 3D culture system, where higher resistance to TQ was observed.
After a further 24 hours, the treatment media were replaced with 100 µl of PBS solution containing MTT (5 mg/ml), and the plate was incubated for 4 hours. After aspirating the medium, the formazan crystals were dissolved in 100 µl of Dimethyl sulfoxide with agitation for 2 hours. The absorbance was measured at 570 nm using a microplate reader. All experiments were done in triplicate.
2.4. HepG2 culture in calcium alginate beads of different sizes (3D culture)
The electrospray technique was employed to prepare alginate beads of different sizes. The system consisted of a high-voltage power supply, a syringe pump, and a 22-G stainless steel blunt needle, as illustrated in Figure 1. Electrospray relies on the application of an electric field to overcome the surface tension at the needle tip, thereby generating droplets smaller than those formed by gravitational dripping. The calcium alginate was dissolved in distilled water at a concentration of 1% (w/v). A 0.1% (w/v) calcium chloride solution served as the gelling bath. A relatively low CaCl2 concentration (0.1% w/v) was used to generate softer alginate beads suitable for cell culture, facilitating nutrient diffusion and maintaining higher cell viability while still allowing stable crosslinking of alginate. 50 mM sodium citrate buffer was used to dissolve (break) the beads. All solutions were sterilized by autoclaving at 121°C. Therefore, 2 ml of calcium chloride solution was added to each well of the 24-well plate. HepG2 cells (5 × 106 cells) were homogeneously mixed in 5 ml of calcium alginate solution and loaded into a syringe fitted with a 22 G needle [34]. A variable positive voltage was applied while the cell suspension was delivered at an adequate flow rate of 0.5 ml/h. At each voltage, 200 µl of the same cell suspension was deposited into the calcium chloride solution. In order to facilitate crosslinking, the plate was left at room temperature for 10 minutes. After that, 0.9% saline solution was used in place of the calcium chloride solution for 15 minutes, and then complete culture medium was added for an additional 15 minutes. The beads were then incubated at 37°C, and 5% CO2 after the medium was changed out for new complete medium [35].
 | Figure 1. The schematic representation of the electrospray apparatus for the formation of alginate beads for cell culture of HepG2 cells. [Click here to view] |
2.5. 3D cytotoxicity assay
HepG2 cells cultured in alginate beads of different sizes were incubated for 4 days at 37°C and 5% CO2. The beads were then exposed to TQ at concentrations ranging from 15.62 to 2,000 µg/ml, consistent with those used in the 2D assay, and incubated for an additional 24 hours. Control beads contained medium without TQ. The crosslinking was reverted using sodium citrate buffer, and the cells were harvested from the wells and then centrifuged to prepare a pellet. The cytotoxicity of cells was assessed immediately after harvesting, in which the cell pellets were cultured for 4 hours in a 96-well plate, followed by the same steps of MTT assay as in 2D cytotoxicity. Control experiments were performed similarly but without adding TQ to ensure the viability of the cells subjected to 3D culture steps.
2.6. Statistical analysis
Statistical analysis was performed using an independent samples t-test for comparisons between two groups (2D and 3D cultures). Data are presented as mean ± SD. IC?? values were calculated using nonlinear regression with a four-parameter logistic dose–response model implemented in GraphPad Prism software. Differences were considered statistically significant at p < 0.05.
3. RESULTS
The impact of the electrical voltage applied to the needle on the size of beads was assessed using an electrospray setup at a solution rate of 0.5 ml/h. The results showed that bead size decreased with increasing voltage (Fig. 2). The impact of voltage and bead size on the viability of HepG2 cells was investigated following the preparation of microbeads utilizing the electrospray setup. The highest cell viability (more than 80%) was observed when 3 kV was applied to the nozzle, which corresponds to the bead size of 2.6 mm, as depicted in Figure 3. Based on the study, 3 kV was the optimal voltage to encapsulate HepG2 in alginate beads using the current electrospray conditions (flow 0.5 ml/h, 22 G nozzle, 4 cm distance). Figure 4 shows the microscopic view of the cells in alginate beads after 4 days of incubation. Microscopic pictures of alginate beads containing HepG2 cells are displayed in Figure 4. Effective bead creation appropriate for 3D cell culture was demonstrated by the spherical, uniformly shaped beads and the efficient encapsulation of the cells within the alginate matrix.
 | Figure 2. Effect of nozzle voltage on alginate bead size at a flow rate of 0.5 ml/h and a 4 cm distance between the nozzle and the calcium chloride solution surface. [Click here to view] |
 | Figure 3. Influence of applied voltage on HepG2 cell viability under a constant flow rate (0.5 ml/h) and a 4 cm distance between the nozzle and the calcium chloride solution surface. [Click here to view] |
 | Figure 4. HepG2 cells cultured in alginate beads observed under the light microscope (10× objective lens) after 4 days of incubation in the humidified cell incubator with 5% CO2. [Click here to view] |
The voltage used in the formulation of alginate beads was 3 kV. Figure 5 illustrates that the proliferation of HepG2 cells was markedly more rapid within the alginate beads than in the conventional cell culture system.
 | Figure 5. Growth pattern of HepG2 cancer cells in 2D versus 3D setting from days 1 to 9. Calculated doubling time for HepG2 cells = 3.56 days in alginate beads and 4.01 days in a conventional culture setting. Data are presented as mean ± SD. Statistical significance was determined using an independent samples t-test for comparisons between two groups (2D and 3D cultures). [Click here to view] |
The influence of TQ therapy on HepG2 cell viability in a 2D environment was assessed after 24 hours of exposure. TQ solution demonstrated a dose-response effect with the concentrations shown in Figure 6. As shown, increasing TQ concentrations to 31.25 µg/ml caused relatively low cytotoxicity to HepG2 cells. Higher concentrations were associated with a significant decrease in cell viability. The high percentage of cytotoxicity was recorded at the highest applied TQ concentration of 2,000 µg/ml.
 | Figure 6. (A). Comparison of HepG2 cell inhibition at different TQ concentrations in 2D versus 3D culture. Percent cell viability is plotted against different TQ concentrations. (B). Comparison of the IC50 value of TQ in 2D versus 3D culture settings. Data are presented as mean ± SD. Statistical significance was determined using an independent samples t-test for comparisons between two groups (2D and 3D cultures). [Click here to view] |
The 3D culture setting was slightly different, whereby the HepG2 cancer cells were grown in the alginate beads for 4 days before treatment with TQ. The same treatment concentrations of TQ were used as shown in Figure 6.
In contrast to the alginate beads culture system, which displayed more viable cells after treatment, the growth of HepG2 cells was considerably inhibited in the 2D cell culture (Fig. 7).
 | Figure 7. Comparison of 2D and 3D culture of HepG2 cells after 4 days of TQ exposure at 2,000 μg/ml. (A–C) Inverted microscopic images (10× objective) of 2D culture setting before treatment; (D–F) Broken beads in 3D culture after 1, 2, and 4 days of incubation; (G–I) Cytotoxicity of TQ in 2D culture. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using an independent samples t-test for comparisons between two groups (2D and 3D cultures). [Click here to view] |
4. DISCUSSION
The impact of the electrical voltage applied to the needle on the size of beads was assessed using an electrospray setup. One of the most critical issues in 3D cell culture is the standardization of alginate bead diameters to obtain reproducible results. For instance, the diameter of alginate beads affects the biological activity of the encapsulated cells. Several studies have shown a significant reduction in cellular interactions when using nanoscale spheroid formulations [36,37]. At the same time, large-scale formulations had also limited the biological activities due to the lack of oxygen and nutrients reaching the core of the sphere [38]. Hence, necrosis can occur with a considerable fraction of cells. Additionally, to achieve a significant level of biological activity with high homogeneity, calcium alginate beads should have adequate diameters with a narrow size distribution [39]. In order to isolate the effect of voltage on bead production, the flow rate, needle gauge, and nozzle distance were kept constant. As a result, the reported optimal voltage (3 kV) particularly relates to the experimental conditions utilised in this study.
The effect of voltage/bead size on the viability of HepG2 cells was demonstrated in this study. Previous studies indicated a substantial improvement in the percentage of viable cells after the application of the electrical field on encapsulated cells [40]. At lower voltages, the bead size was larger, resulting in hypoxia and a lack of nutrients in the bead center. On the other hand, higher voltage values may induce more stress to the cells, resulting in reduced cell viability.
The study has shown the higher proliferation of HepG2 cells in the alginate beads, which was significantly higher compared to the conventional cell culture system. This property is attributed to changes in cancer cells’ cytoskeleton, intercellular interactions, and the different arrangements of surface receptors in the 3D system, leading to enhanced proliferation of cancer cells [19,24,41].
There is a considerable difference in the sensitivity of cancer cells in the two culture systems at concentrations close to the IC50 value of TQ in the 2D setting. The IC50 in 2D cell culture was 384 µg/ml, whereas for 3D culture, the IC50 value was 916 µg/ml. The higher IC50 of 3D alginate hydrogel culture demonstrated an increased resistance against TQ when compared to the 2D cell culture. The use of a 3D alginate bead culture technique may have contributed to the study’s comparatively higher IC50 values, since cells grown in 3D environments frequently exhibit greater resistance to anticancer drugs when compared to traditional 2D monolayer cultures. The effective concentration of TQ that reaches the encapsulated cells in 3D alginate bead systems may be limited by drug diffusion barriers, which could explain the variations in 2D and 3D culture responses.
As mentioned earlier, the monolayer systems demonstrate an exaggerated treatment response due to the high surface area of exposure to treatment, in addition to the lack of cellular interactions. In this study, alginate beads were dissolved with sodium citrate prior to the MTT experiment in order to assess the vitality of encapsulated HepG2 cells. Because diffusion barriers inside the alginate matrix may restrict reagent access, this step guaranteed that cells were exposed to the MTT reagent uniformly. The cells were already subjected to 3D culture conditions and TQ therapy while encapsulated, even though this approach momentarily removes the cells from the 3D environment during measurement. In-bead viability tests could be used in subsequent research to better maintain the 3D culture system’s structural integrity.
Accordingly, the culture of HepG2 cells in 3D alginate hydrogel in vitro demonstrated significant resistance to TQ compared to the 2D setting due to the multicellular architecture of the cell mass in the alginate beads that mimics the conditions found in solid tumors [41–44]. The obtained viability data indicate that the encapsulated HepG2 cells were viable under the experimental conditions used, despite the relatively large bead diameter (about 2–3 mm) potentially influencing nutrition and oxygen diffusion within the beads.
Compared to traditional 2D cultures, the alginate bead-based 3D culture method offers a more physiologically appropriate environment. Increased cell–cell and cell–matrix interactions, which may affect cellular activity and medication responsiveness, are among the features of the tumour microenvironment that this model may replicate.
Recent studies have also highlighted the importance of advanced 3D culture models in drug screening [45,46].
Therefore, the 3D systems offer a better model and a closer representation of the in vivo systems that may help in providing a better understanding of cell interactions [47].
The demonstrated cytotoxicity of TQ in HepG2 liver cancer cells could be attributed to the reported anticancer properties of TQ, including induction of cell cycle arrest of cancer cells in G0-G1 and G1 phases [48].
The particle size challenged the culture of HepG2 cells in a 3D alginate setting, as the mass transport was affected by the tumor cells at the periphery, depriving the cells in the center of nutrients, oxygen, and CO2, resulting in necrotic cells in the center [49]. Consequently, the monolayer systems are more homogeneous and not limited by mass transport.
5. CONCLUSION
The 3D culture of HepG2 cells on calcium alginate provides a more representative model of cancer cells to study the cytotoxic activity of anticancer drugs. It has been found that the growth of HepG2 cells was inhibited by TQ in 3D culture. In calcium alginate beads, inhibition of cell growth following TQ treatment was lower compared to the 2D culture growth at the same concentrations. Calcium alginate offers several advantages, including biocompatibility, biodegradability, and low immunogenicity, making it suitable for use as a 3D scaffold for cultured cells. This 3D culture system provides preliminary indications that could be further developed for investigating the anticancer compounds.
6. ACKNOWLEDGMENTS
The authors would also like to acknowledge the support of the Scientific and Technical Research Center in Physicochemical Analysis, Bou-Ismail, Tipaza, Algeria; the University Putra Malaysia; and the International Islamic University Malaysia.
7. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
8. FINANCIAL SUPPORT
There is no funding to report.
9. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
10. ETHICAL APPROVALS
The study involved commercially available established cell lines (HepG2) and did not involve human subjects or animal experiments. Therefore, ethics committee approval was not required according to institutional guidelines.
11. DATA AVAILABILITY
The data that support the findings of this study are available from the corresponding author upon reasonable request.
12. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
13. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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