Page 567 - IJB-10-3

International Journal of Bioprinting Bioprint micro breast cancer

2.1. Cell culture To evaluate the impact of the bioprinting process on cell
In our study, we primarily selected the SUM149 cell viability, we assessed the viability of cells before and after
line due to its aggressive nature and origin from ductal- bioprinting in both the bioink and the bioprinted tissues.
type breast cancer, providing a more direct objective for This assessment was conducted using the CellTiter-Glo®
mimicry. While SUM149 and MDA-MB-231 are both 3D Cell Viability Assay (Promega Corporation, USA), in
triple-negative cell lines, we limited the use of MDA- accordance with the manufacturer’s instructions. Briefly,
MB-231 and MCF-7 to the drug prediction segment of the CellTiter-Glo® 3D reagent was added directly to each
our study. This decision was informed by their relevance sample. To ensure complete cell lysis and optimal assay
to corresponding clinical trials and previous studies, performance, the samples were gently mixed and then
facilitating easier comparisons. Recognizing the challenges incubated, allowing the luminescent signal to stabilize.
in harvesting breast-originated fibroblasts, we opted for Luminescence was quantified using a luminescence
normal human lung fibroblasts (NHLFs) to model cancer- counter, with the intensity of the luminescent signal
associated fibroblasts (CAFs) for breast cancer, owing to serving as a readout for cell viability within the 3D
the ectodermal origins and glandular structures shared structures. For comparative analysis, all data were
by both the breast and lung. Lastly, we utilized human normalized to the average luminosity observed prior to the
umbilical vein endothelial cells (HUVECs) to model bioprinting process.
microvasculature, given their wide availability and proven 2.3. Cell labeling using CellBrite dyes
efficacy in such applications. Additionally, we incorporated CellBrite Blue, Green, and Red Cytoplasmic Membrane
the Jurkat cell line to represent T-cell interactions in the Dyes (Biotium, USA) were utilized to label distinct cell
tumor microenvironment. types in bioprinted PMCaTs, as per the manufacturer’s

Three human breast cancer cell lines were utilized: recommendations. Cells, upon reaching 70–80%
SUM149 cells cultured in Ham’s F-12 medium, and both confluence, were washed with PBS, treated with a serum-
MCF7 and MDA-MB-231 cells in Dulbecco’s Modified free medium, and subsequently incubated with a 5× dye
Eagle Medium (DMEM), all supplemented with 10% solution at 37°C for 20 min. After washing off excess dye
fetal bovine serum (FBS) and 1% penicillin-streptomycin. and a subsequent 30-min incubation in fresh medium, the
NHLFs were grown in fibroblast basal medium with cells were visualized under a fluorescence microscope to
fibroblast growth kit components as per Lonza’s confirm successful labeling. Analysis of the labeled cells
specifications, and HUVECs were grown in endothelial within the bioprinted tissues was conducted using image
basal medium-2 (EBM-2) enhanced with the EGM-2 analysis software, with quantification based on the intensity
SingleQuots kit, according to Lonza’s guidelines. Jurkat and distribution of each fluorescent label. Experiments
cells, a T-cell line, were cultured in RPMI 1640 medium were carried out in triplicate.
with the same supplements. All cells were maintained in a 2.4. Bioprinter design and mechanism
humidified atmosphere at 37°C and 5% CO₂, with media The DVDOD 3D bioprinting system has been detailed in
refreshed every 2–3 days. Adherent cells were passaged prior publications, 13,14 and is also depicted in Scheme 1A
using a 0.05% trypsin-EDTA (ethylenediaminetetraacetic and B. Our bioprinter distinctively utilizes a direct control
acid) solution at 70–80% confluency, while suspension- mechanism whereby the bioink droplet volume is regulated
growing Jurkat cells were passaged by dilution. Rigorous by a linear actuator. This method contrasts existing
standards were upheld throughout to prevent cross- systems, which commonly adjust fluid-valve durations or
contamination and ensure cell line purity. the bioink’s applied pressure.
2.2. Cell viability assessment using calcein The bioprinter is constructed with a three-axis linear
AM staining motion gantry equipped with multiple linear dispensing
Cell viability within the bioprinted PMCaTs (n = 3) was units, each designed to enhance the precision of the
assessed using calcein AM (Thermo Fisher Scientific, bioprinting process. Each of these units possesses a syringe
USA). Microtissues were first rinsed with phosphate- driven by a linear actuator, enabling the volumetric
buffered saline (PBS) to remove any residual culture manipulation of bioink dispensed. Upon the introduction
medium and then incubated with a 2 μM calcein AM of bioink into the air-driven dispensing nozzle, pulsed air
solution at 37°C for 30 min. After washing with PBS, the is injected, facilitating the expulsion of the determined
tissues were immediately visualized under a fluorescent bioink volume. These dispensing units harmoniously
microscope. The observed green fluorescence, resulting control droplet locations and their volumes on the
from the conversion of calcein AM to fluorescent calcein substrate. To ensure optimal bioprinting conditions, the
in live cells, signifies viable cells. printer is equipped with temperature and humidity control

Volume 10 Issue 3 (2024) 559 doi: 10.36922/ijb.2911