Page 570 - IJB-10-3

International Journal of Bioprinting Bioprint micro breast cancer

life for elution of PKH26 from labeled cells exceeds 100 normalized against the control group to determine relative
days, this enabled the tracking of all cells in PMCaTs cell viability.
throughout the metastasis simulation. Bioprinted PMCaTs
were strategically positioned atop a nylon mesh and each 2.14. Statistical analysis
microtissue was firmly attached to the mesh using a small Data are reported as mean ± standard deviation (SD).
droplet of fibrin glue, ensuring its stability during the The independent t-test was employed to determine the
observation period. Over a designated timeframe, two differences between two distinct groups. For comparisons
key metastatic behaviors were observed: (i) the release of across multiple groups, analysis of variance (ANOVA) with
small cellular aggregates from the original microtissue, the Tukey post-hoc test was used. A significance level was
emulating the initial step of in vivo cancer metastasis established at p < 0.05.
where tumor cells detach from the primary tumor, and
(ii) the initiation of new tumor growth at a distance (>5 3. Results and discussion
mm) from the original micro-cancer tissue. Remote tumor 3.1. Viability and structure of printed micro-cancer
growth was identified as a metastatic event, especially tissues under fluorescent microscopy
given the lack of a fluorescent migration path linking it to Our bioprinting technique proficiently produces PMCaTs
the original tissue. with three distinct cell types (Figure 1A–D). Cancer
cells, fluorescently labeled in red, mimic natural cancer
2.13. Cell proliferation upon alisertib treatment
nests with their sub-grouped arrangement. Fibroblasts,
2.13.1. Drug preparation fluorescently labeled in blue, depict the role of CAFs in
Alisertib was dissolved in dimethyl sulfoxide (DMSO) to genuine tumor contexts. Additionally, the inclusion of
create stock solutions. From this stock, it was diluted with endothelial cells, fluorescently labeled in green, simulate
culture medium to achieve the specified concentrations: the microvascular system, underlining the intricacy of our
2, 20, 100, and 200 μM. For control experiments, an bioprinting approach. Notably, the PMCaTs maintained
equivalent volume of DMSO, matching the highest alisertib consistent viability over 4 weeks (Figure 1E–G), illustrating
concentration, was used to discern any potential effects of the robustness of our technique. This sustained vitality not
the solvent alone on cell viability. only attests to the method’s efficiency but also suggests
its suitability for prolonged research and therapeutic
2.13.2. 2D cell viability assay using CellTiter-Glo evaluations. Integrating pivotal elements of the cancer
MDA-MB-231 and MCF-7 cells were seeded into 96- microenvironment like CAFs and microvasculature,
19
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well plates at a density of 5000 cells/well and incubated alongside detailed tissue architecture and sustained
overnight to ensure complete attachment. Following viability, positions this approach as a valuable tool for
attachment, the cells were treated with concentrations of comprehensive oncological studies.
0, 2, 20, 100, and 200 μM of alisertib (n = 3 per condition)
and incubated for an additional 48 h. Subsequently, 100 μL In our current project, the data corroborate the efficacy
of CellTiter-Glo reagent was added to each well, ensuring of the DVDOD technique in maintaining cell viability,
thorough mixing for cell lysis. The plates were then set aside aligning with our prior observations. Notably, cell viability
at room temperature for 10 min, allowing the luminescent remained virtually unchanged during the bioprinting
signal to stabilize. Luminescence was quantified using a process (100.0 ± 6.03% before bioprinting vs. 94.6 ± 8.24%
plate-reading luminometer. after bioprinting, with data normalized to pre-bioprinting
values, p > 0.05) (Figure 1H). This outcome was attributed
2.13.3 Micro-cancer tissue bioprinting and cell to the application of air pressures in a non-continuous
viability assay using CellTiter-Glo 3D mode, ensuring cells were exposed to pressure only during
PMCaTs were generated using the protocol detailed in the fluid-driving phase. This approach contrasts with other
section 2.5. Bioprinting process. They were placed into DOD techniques where cells are subjected to continuous
the wells (n = 3 per concentration) of a 96-well plate, air pressure. We have tested various hydrogels, including
incubated, and treated with concentrations of 0, 2, 20, 100, collagen, alginate, and fibrin, in different concentrations
and 200 μM of alisertib for 48 h. At the end of the assay, and volumes. Unlike other DOD techniques, the DVDOD
100 μL of CellTiter-Glo 3D reagent was introduced to each method maintains a more constant droplet velocity exiting
well. The plates were subjected to agitation on an orbital the nozzle, as it does not rely on altering pressure to
shaker for 5 min. This was followed by a 25-min incubation change the droplet size of bioink. This technique, which
at room temperature to stabilize the luminescent signal. applies minimal pressures only during the fluid-driving
Luminescence intensities were captured using a plate- phase, stands in stark contrast to the continuous pressure
reading luminometer. Luminescence readings were then application seen in valve-based bioprinting. HUVECs

Volume 10 Issue 3 (2024) 562 doi: 10.36922/ijb.2911