Fig 1.
Cell culture using simple to complex three-dimensional bio-printing models.
Schematic illustration of the 3D bio-printing process and image of the printing setup and constructs. Platform designed using a computer program. The overall size is 1 × 1 × 0.01 mm, and the order is x, y, and z-axis (left image). Top view of 3D bio-printing construct used GelMA demonstrating the porous platform of the scaffold (middle, right images).
Fig 2.
UV crosslinking condition for 3D culture model.
(A) After adding 1 ml to the vials, the UV crosslinking time condition was confirmed. The times were fixed at 0, 30, 60, 90, 120, and 180 s, and a 365 nm UV lamp was used. (B) The pattern was printed on a 60-mm plate using GelMA. After crosslinking at the conditions provided in (A), complete media was added and samples were incubated for 1 day in a 37°C, 5% CO2 incubator. (C) The shape of the structure was measured after 90, 120, and 180 s. The intensity force at which the structure was destroyed under the conditions of constant distance (1.225 mm) and time (0.7 s) was measured. (D) The GelMA structure and GelMA/bladder cancer cells mixture was printed and crosslinked. After being placed in complete media, the GelMA structure was confirmed with a microscope, and the structure was maintained after incubation for 5 days. (E) To investigate the effect of crosslinking time on the cells, we performed live/dead staining. After 1 day of culture, the printed mixture of GelMA and cells was treated with Calcein-AM (2 μM) and EthD-1 (4 μM) and examined by fluorescence microscopy. The shape of the structure was examined. (F) The different layers of scaffold in the Z-direction. The constructs were printed at different heights of 0.03, 0.08, 0.1, and 0.15 mm with only GelMA. Some of the structure was maintained even after 1 day in the complete media.
Fig 3.
Morphological changes in the cells of printed constructs and comparison of 2D and 3D cell proliferation.
(A) To confirm the morphology of the bladder cancer cells, 1 × 105 5637, T24 cells were seeded on a 60-mm plate dish, stained with fluorescence markers, and observed with a microscope for 5 days. For 3D cell culture, 1 × 105 cells were mixed in 1 ml solution and then the construct was printed. The cells (1 × 105) were used in a total of 100 μl solution to print one structure. Live/dead staining in the 2D and 3D cell culture environment confirmed cell viability. Cells were stained with Calcein AM (2 μM) and EthD-1 (4 μM) and observed after 1, 3, and 5 days with a fluorescence microscope. (B) The 5637 and T24 human bladder cancer cells were seeded on 60-mm plates for 1, 2, and 3 days. Cell proliferation was determined by the CCK-8 assay in bladder cancer cells. * p < 0.05, ** p < 0.01, ratios were compared between 2D and 3D cultures on each day. Data are the mean ± SEM (n = 6).
Fig 4.
Drug treatment of bladder cancer cells in 2D and 3D environments.
(A) Effect of treatment with rapamycin (inhibitor of mTOR) and Bacillus Calmette-Guérin (BCG) on viability of bladder cancer cells. Cells were treated with indicated concentrations of rapamycin (1 μM, BCG, 30 MOI) for 1, 2, and 3 days. Absorbance was measured at 540 nm and cell viability was determined by the MTT assay. * p < 0.05, ** p < 0.01, ratios were normalized to each untreated group. Data are the mean ± SEM (n = 6). (B) Western blot analysis of 5637 and T24 bladder cancer cells cultured in 2D and 3D compared the expression of mTOR-mediated p70s6K and 4E-BP1 signal pathways. Cells were treated with rapamycin (1 μM) for 24 h and then the cells were lysed. Western blotting results showed a reduced level of expression in cells in the 2D environment due to treatment with rapamycin. GAPDH was used as a loading control. A representative result from three experiments is shown.
Table 1.
The concentrations of cytokines in 2D and 3D bladder cancer cell environments after BCG treatment.
Fig 5.
Cell-to-cell marker protein expression in the 2D and 3D cell culture environment.
(A), (B) Induction of release of the epithelial-mesenchymal transition (EMT) mechanism protein in bladder cancer cells treated with TGF-β1. After 72 h, the supernatant of the 5637 and T24 bladder cancer cell culture media was separated, and e-cadherin and n-cadherin were measured by sandwich ELISA. * p < 0.05, ** p < 0.01, ratios were compared between the 2D- and 3D-cultured TGF-β1-untreated group and the 2D- and 3D-cultured TGF-β1-treated group. Data are the mean ± SEM of three independent experiments.
Fig 6.
Hypothetical schema of comparison of drug resistance effect in 2D and 3D environments due to cell-to-cell interaction.
The 2D cell culture environment and 3D cell culture environment reacted to rapamycin and BCG treatment, and there were strong differences. The mTOR signal pathway was more highly blocked by rapamycin in 2D than in 3D. The secretion of IL-6, IL-12, and IFN-γ, which are antitumor cytokines, due to BCG was also increased more in 2D than in 3D, resulting in an enhanced antitumor effect on bladder cancer cells. This indicates that our model confirms the secretion of E-cadherin and N-cadherin and shows the difference in drug resistance according to the difference in the intensity of cell-to-cell interaction. The results provide a rationale for evaluating drugs using the 3D cell culture environment.