1. Introduction

Tumors consistently develop as highly complex structures composed of genetically altered cells, together with fibroblasts, endothelial cells, pericytes, and inflammatory cells embedded in the extracellular matrix (ECM) [1]. Employing different mechanisms, cancer cells are almost invariably resistant to a variety of anticancer drugs [2]. Multidrug resistance in cancer is frequently associated with the overexpression of active transporters that act as drug efflux pumps [3, 4]. Tissue structure determines the growth rate of a tumor and the response to anticancer drugs; there are studies indicating that three-dimensional (3D) tumor models are better representatives of cancer tumors in vivo than two-dimensional (2D) ones [5, 6]. To create and recapitulate cellular in vivo conditions, biomaterial-based substrates capable of generating precise patterning of cells for 2D or 3D cell culture systems and for controlling microenvironmental stimuli at the single-cell level (approximately 10 μm) in microsystems are crucial [7]. These substrates have served as versatile tools to explore fundamental cell biology, tissue engineering, and drug development, and thereby have become key components in a wide range of applications [8]. In past few yeas, a 3D cancer cell system has been employed to study the charactristics of cancer cell behaviour. Thus, various techniques for patterning cells in 3D tissue cultures have demonstrated the ability to direct cells onto selected areas of a substrate, including soft lithography or microcontact printing, microstructure- or microfluidic-based systems, active control of cell attachment or suspension by physical forces, and layer-by-layer assembly system [7, 9–13].

In this study, the 3D cell chip, which has an open-cell-structure dependent on volume and allows movement anywhere in the three degrees of freedom (x, y, z), is ideally suited for the growth of a living cell. Specifically, we have found that two-photon stereolithography is an effective method to fabricate 3D polymer microstructures. Our system establishes conditions that are closer to those found in nature. In the present study, we have developed a novel technology able to produce user-friendly architecture devices that can be readily employed for 3D cell growth simulation of in vivo tumors. Moreover, our results demonstrate that the developed 3D cell chip can control the growth of a cell depending on the size of the cell room, and can easily form a tumor spheroid. Also, this system controls the growth of the tumor depending on the 3D cell chip volume. Furthermore, we investigated the formation of Du 145 cell spheroids, acting as an in vivo tumor model, to evaluate the 3D cell chip for tumor observation.

2. Experimental

2.1 Fabrication of the 3D cell chip

A 3D pyramid cell chip was fabricated using two-photon stereolithography (TPS) [12–19]. A schematic diagram of the TPS process setup is shown in Fig. 1. A photo-polymerizable resin (SU-8; Microchem Corp.) was dropped on a cover-glass plate fixed on an XYZ piezoelectric stage (800 μm × 800 μm × 400 μm). A femtosecond laser (mode-locked Ti: Sapphire laser) was used for two-photon absorption and was tightly focused in the resin using an oil-immersion objective lens (N.A. of 1.3, 100 × magnification). The laser focus was fixed at a given position, and the cover-glass plate was fixed onto the jig. The specimen was moved three-dimensionally according to the 3D laser scanning data using the XYZ piezoelectricstage with a resolution of 0.1 nm. A high-magnification charge-coupled device (CCD) camera was used for the precise control of the laser focus and for real-time fabrication monitoring (see Fig. 1).

 

Fig. 1 Schematic diagram of the process for 3D cell chip. Manual stages control the X, Y, and Z positions of the piezoelectric stages of the specimen. The piezoelectric stages control the X, Y, and Z positions of the specimen over the range of 800 μm × 800 μm × 400 μm with the resolution of 0.1 nm. A femtosecond laser (mode-locked Ti-sapphire laser) was used as a laser beam source; the laser has the wavelength of 780 nm, ultrashort pulse duration below 100 fs, and the repetition rate of 80 MHz. A λ/2 plate and polarizing beam splitter were used to control the laser power.

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SU-8 resin was employed for the fabrication of the 3D pyramid cell chip. The SU-8 resin used for the TPS process was composed of a two-photon absorbing (TPA) material and a photo-acid generator (PAG). Two-photon absorption leads to the generation of photo-acid (H⁺) in the SU8 photoresist. After photocuring the photoresist is heated at 95°C for 15 min leading to formation of hardened polymeric structures in the exposed areas through cationic polymerization. The uncured area of the SU-8 resin was removed using the propylene glycol monomethyl ether acetate (PGMEA) solution.

2.2 Du145 prostate cancer culture in the 3D cell chip

For the cell analysis, a cell chip chamber (1 cm × 1 cm × 0.5 cm) was fabricated via the fixing of a plastic chamber (Lab-Tek R). 2D and 3D pyramid cell chips have the same size chamber, but the 3D cell chip has 4 pyramid structures. Du145 cells were purchased from the Korea Cell Line Bank (Seoul, Korea). Human prostate cancer Du145 cells were cultured in RPMI 1640 medium (Welgene) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 200 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified chamber with 5% CO₂ at 37°C. The external surface of each sample was seeded with 1 ml of a cell suspension at the seeding density of 4 × 10⁴ cells/ml. The cell density enabled the quantification and imaging of the cellular processes before confluence. The cells were treated either with vehicle alone or with the specified concentration of mitoxatrone dissolved in deionized water. The tumor population was determined using SEM (S-800 Hitachi, Japan) and inverted microscopy, and was calculated by dividing the average number of cell by the total number of spheroids over a 14-day period. The cultured cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich) at a final concentration of 0.1 μg/ml in the phosphate buffered saline (PBS) at 30°C for 15-30 min. The total cell number/pyramid structure was analyzed using Image J software (image.J.nih.gov/ij), and the cell units containing a tumor were counted as positive. Surface morphologies were analyzed by SEM.

2.3 Immunofluorescence staining

The Du145 cells were plated on the 3D cell chip and grown until the cells reached 60–70% confluence. Cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 for 1.5 hours, and then blocked with 5% bovine serum albumin (BSA). The primary antibodies used were F-actin. The secondary antibody was labeled with fluorescein isothiocyanate (FITC, green; Invitrogen). The cells were viewed and photographed using either a Zeiss Axio Imager M1 microscope (Carl Zeiss MicroImaging) or a confocal Leica-SP2 UV (Leica Microsystems). Images were compiled using Adobe Photoshop.

2.4 Cell viability assay

The effects of oleanolic acid on cancer cell proliferation and cell viability were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Genetrone, Korea) assay. The cells were seeded in a well plate at the density of 4 × 10⁴ cells/well and incubated for 24 h to allow for attachment. After mitroxanthrone (100, 200, 300, 400, and 500 μg/ml deionized water) treatment, the cells were incubated for 24 h at 37°C. Absorbance was measured at 540 nm using a microplate reader (Asys UVM 340, Biochrom Ltd, Cambridge, UK) after the addition of the MTT solution (5 mg/ml) for 4 h.

3. Results and discussion

3.1 Fabrication of the 3D cell chip

We designed a 3D structure for the fabrication of the 3D cell chip for the trophoblast stem (TS) cell culturing. As shown in Fig. 2, the 3D pyramid structure was a three-floor structure composed of 116 individual compartments with 64, 36, 16 compartments on the first, second, and third floor, respectively. The size of each compartment was 12 μm × 12 μm × 12 μm. As the 3D structure was fabricated with thin connected wires, each compartment of the structure was empty, and tumor could be cultured in 3D inside the compartment.

 

Fig. 2 The SEM image of the 3D cancer cell chip fabricated on the ITO glass. The structure consists of three floors. The dimensions of each unit compartment are 12 μm × 12 μm × 12 μm. The first floor consists of 64 compartments, the second floor consists of 36 compartments, and the third floor consists of 16 compartments.

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For the general two-photon process, a 3D structure is conventionally fabricated using the conventional layer-by-layer accumulation method, which involves the layer-by-layer accumulation of 2D scanning paths of a laser beam [12–17]. However, when using the layer-by-layer accumulation method to fabricate the supporting pillars of the 3D pyramid structure, a long fabrication time is necessary, and the fabrication accuracy is limited. Therefore, in the present study, we fabricated a 3D structure using a new laser scanning technique employing the multi-directional laser scanning (MDLS) method. The MDLS method is a technique which improves the efficiency of the laser scanning process over the layer-by-layer accumulation method. As shown in Fig. 2, the MDLS method involves laser scanning on the XY plane for the fabrication of the base and laser scanning in the Z-direction for the fabrication of the pillars. Because x, y, and z piezoelectric stages are controlled simultaneously for the multi-directional laser scanning method, the 3D microstructure can be fabricated by continuously scanning at the x-, y- and z-axes. The use of the multi-directional laser scanning method ensures a high-precision structure with a short fabrication time as compared to that of the conventional method.

In addition, when the 3D pyramidal structure was developed using the PGMEA solution, it was observed that the structure became twisted near the third floor due to the flow of the solution. To prevent this distortion, the structure should be fabricated as a high strength structure up to the third floor. Thus, each floor requires a different laser scanning speed to control its strength. To create the first floor, we used the laser scanning speed of 30 μm/s, whereas for the second and third floors, the speed was adjusted to 10 μm/s. As a result, the second and third floors were thicker than the first floor. Figure 2 shows the 3 pyramid–like structure of the 3D cell chip. Thus, the two-photon stereolithography process was demonstrated to be an effective method for fabricating 3D polymer microstructures. The multi-directional laser scanning method was used for the precise fabrication of open-cell structures. This process offers remarkable advantages as a direct, high-resolution 3D polymer patterning process that can be used to create precise 3D microstructures for cell culturing applications.

3.2 Cell proliferative characteristics of the 3D cell chip

The 3D cell chip fabricated by two-photon stereolithography represents all-3D layered structures with core-only and core-shell shapes. When observing the cell morphology, the Du145 cells cultured on smooth substrates displayed random cell spreading. As mentioned above, the medium placed on the surface of the 3D cell chip adopted a pyramid shape, preserving the high relative humidity (95%) of the cell culture incubator and preventing the evaporation and the subsequent reduction of the chamber volume. When the cells were seeded onto the 3D cell chip, they were first randomly distributed within the medium. In the following hours, the cells settled and accumulated at high density in a highly localized region, namely, the contact region between the space of the pyramid and the surface of the pillars. The SEM image revealed spheroid morphology in the appearance of the Du145 cells cultured on the 3D substrates (Fig. 3). The plan-view image shows tumor in the 3D cell chip. Du145 cells cultured on the 3D cancer cell chip retained high levels of cell viability, possessed excellent cellular morphology, and showed enhanced function compared to their counterparts cultured on a 2D plate. Also, we observed that the Du145 cells showed a flat, well-spread morphology with small microvilli on their surface in 2D plate. Cells in a living organism are surrounded by other cells and the extracellular matrices (ECM). To mimic this environment, several laboratories have shown that cell cultures in a 3D space are more physiologically relevant, as compared to the cells cultured as monolayers [7, 20]. Herein, we report the development of a novel device that enables 3D cell growth in vitro. Several unique aspects of the design of this technology are essential to the device’s development as a research tool and intended application, and we have overcome various obstacles to achieve a working prototype. The system demonstrated sufficient size uniformity among cells, better than the one of the 2D plate control.

 

Fig. 3 The SEM images of prostate cancer cells on the fabricated 3D cell chip and 2D plate

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Cells were cultured in a 3D fabrication structure for 3 days and stained with F-actin (Fig. 4). The actin cytoskeleton of 2D plate was reorganized with the formation of F-actin rich protrusions at the leading edge of an invasive tumor, where the cell shape changed from a cuboidal shape to a motile spindle shape (Fig. 4(a)-4(c)). Additionally, the actin fiber was accompanied with tumor in the 3D cell chip (Fig. 4(d)-4(f)). These processes are involved in promoting all aspects of tumor growth, such as cell proliferation, adhesion and dispersion, and host defense evasion [21]. Higher-magnification imaging of individual cells grown in 3D revealed a significantly greater number of spheroids than that observed for cells grown on 2D surfaces. Additionally, 3D cancer cells adhered to each other and formed aggregates, whereas unaggregated cells on the 2D ITO plate were easily washed out with fresh medium. This system is proposed to have potential for high-throughput drug testing and to be compatible with high-content analysis, due to its ability to be imaged in multiple dimensions (X, Y and Z). A more immediate benefit of 3D lithographic cancer cells might be the ability to make tissue samples that could be used to accurately test drug compounds for toxicity in humans, without the need for animal testing [22].

 

Fig. 4 Confocal microscopic image of F-actin in the prostate cancer cells (Du 145 cells) in the 2D plate and the 3D cell chip. Immunofluorescence images of F-actin in Du145 cells cultured for 5 days in the 2D plate (a-c) and the 3D cell chip (d-f). Confocal microscopy images of cells with F-actin staining (green) using a FITC-conjugated antibody. Nuclei (blue) stained with 4,6-diamidino-2-phenylindole (DAPI). Merged image (f) of F-actin and the DAPI staining. The images were acquired using a Zeiss Axioimager M1 microscope or Leica-SP2 UV confocal microscope using the LSM imaging software program (Carl Zeiss, Jena, Germany) (scale bars: 10 μm).

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3.3 Antitumor activity of mitroxantrone in a 3D cell chip

To investigate the effects of the anticancer drug such as mitroxanthrone on the viability of Du145 prostate cancer cells, we treated cells with 100, 200, 300, 400 and 500 µg/ml mitroxanthrone for 24 h incubation, and assessed cell viability with the MTT assay (see Fig. 5). Interestingly, when comparing the potency of mitroxanthrone in 2D and 3D cultures at the same seeding density, we observed statistically significant differences in the half maximal inhibitory concentration (IC₅₀) values. Our results demonstrate that mitroxanthrone cytotoxic effect increased in the dose-dependent manner in 2D and 3D cultures. Interstingly, the IC₅₀ values of mitroxanthrone in the 2D plate were at the concentration of 345.65 µg/ml. By contrast, the IC₅₀ value of mitroxanthrone in the 3D cell chip was not detected by the system. Previous studies reported that doxorubicin entered spheroids in a 3D culture system much more slowly than an equivalent number of monolayer cells [23]. This demonstrated that the cancer cells in the 3D cancer cell chip provide a multicellular resistance model that mimics the chemotherapy resistance often found in solid tumors in vivo [22, 23].

 

Fig. 5 Anticancer drug-induced apoptotic cell death in the Du145 cells in the 2D plate and the 3D cell chip. Cytotoxicity of mitroxanthrone in the Du145 cancer cells as determined using the MTT assay. The effect of mitroxanthrone (100, 200, 300, 400, and 500 μg/ml) on cell viability compared to the control group. *p<0.05 vs. 2D plate control.

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Generally, various types of biological activities have been reported for mitroxanthrone, including hepatoprotective, anti-fertility, anti-carcinogenic, anti-inflammatory, and chemopreventive effects [24, 25]. Also, the phenomenon of cell spheroids displaying elevated chemoresistance to anticancer reagents has been attributed to several mechanisms, including a decreased penetrance of anticancer drugs, increased pro-survival signaling, and/or upregulation of genes conferring drug resistance [26]. In addition, drug resistance plays a major role in the failure of certain chemotherapeutic agents for the treatment of solid tumors. Various structures of spheroids have shown drug resistance and are also influenced by the effects of different oncogenic molecule expressions [27, 28]. Therefore, our results suggest increased chemoresistance of cells in 3D models to anticancer drugs.

4. Conclusion

Our 3D cell chip demonstrated that cancer cells of a uniform size could be cultured in a less-expensive, faster, and easier manner than the one afforded by manual methods. The first advantage is the uniformity of the structures used to generate specific cell types. Second, the generated cell chips of spheroids are easily accessible for experimentation on single spheroids, making this method compatible with high-throughput drug testing. Since these 3D tumor are also generated in uniform fabricated 3D structure cell chips, large numbers of morphologically homogenous spheroids can be easily produced, which is ideal for high-throughput investigations of the efficacy vs. toxicity of drugs, gene expression in spheroids, and numerous other cellular and biochemical assays.