Page 569 - IJB-10-3

International Journal of Bioprinting Bioprint micro breast cancer

2.6. Detection of hypoxic regions in bioprinted in a collagen I hydrogel at 2 mg/mL concentration, and
micro-cancer tissues subsequently cultured in a complete medium. For drug
Hypoxia in PMCaTs was evaluated using the efficacy studies, we exposed the embedded PMCaTs to
Hypoxyprobe™-1 Omni Kit (Hypoxyprobe, Inc., USA). 0, 2, 20, 100, and 200 μM (n = 3 per concentration) of a
Pimonidazole in the kit binds to thiol groups in hypoxic 5-fluorouracil (5-FU) and then imaged them daily for 3
cells (oxygen levels <10 mm Hg) for subsequent days using a phase contrast microscopy (Nikon, Japan).
immunohistochemical detection. PMCaTs (n = 3) were The invasion dynamics under drug treatments were
treated with 200 μM pimonidazole hydrochloride for 3 benchmarked against untreated controls. Images of the
h (omitted for the negative control), then fixed with 4% invasion of cancer cells into the surrounding collagen
paraformaldehyde, permeabilized, and blocked with matrix were binarized, and the ratios of the invaded area
bovine serum albumin (BSA). Subsequent incubation to the original microtissue area were computed as the
with fluorescein isothiocyanate (FITC)-conjugated anti- primary metric. This analysis was conducted using ImageJ
pimonidazole antibody enabled visualization under a (https://imagej.nih.gov/ij/; National Institutes of Health,
confocal microscope at excitation/emission wavelengths of USA). For this assay, a 3-day endpoint was established
495/519 nm. based on preliminary empirical evidence that such a
timeframe was adequate to observe the differential effects
2.7. Hematoxylin and eosin staining of of the drug concentrations tested. The assay duration was
bioprinted tissues limited to 3 days to prevent cell invasion from extending
PMCaTs were subjected to hematoxylin and eosin (H&E) beyond the visual field of the microscope, which would
staining for histological analysis. Following fixation in impede accurate assessment and measurement.
10% neutral buffered formalin, the tissues underwent
dehydration, xylene clearing, and paraffin embedding. 2.10. Drug penetration modeling
Sections of 5 µm were prepared using a microtome and The penetration of drug compounds into the PMCaTs (n =
subsequently stained with hematoxylin for nuclei and 3) was studied using a model drug, Alexa Fluor 488-labeled
eosin for cytoplasm and extracellular matrix. dextran (70,000 MW, Invitrogen, USA). The bioprinted
PMCaTs were submerged in PBS containing Alexa Fluor
2.8. Confocal analysis of microvascular development 488-labeled dextran. The penetration and distribution of
in bioprinted tissues the Alexa Fluor 488-labeled dextran within the bioprinted
PMCaTs were composed of cancer cells labeled with PMCaTs were visualized using multiphoton microscopy
CellBrite Red, NHLFs with CellBrite Blue, and HUVECs. (Olympus, Japan).
These tissues were embedded in a collagen I hydrogel
(Corning, USA) prepared at a concentration of 2 mg/mL, 2.11. Modeling T cell-based immunotherapy
in line with the manufacturer’s guidance. After a vascular Before bioprinting, all cells designated for the micro-cancer
development duration of 3 days, the tissues were fixed with tissue, including SUM149 cells, NHLF, and HUVECs,
4% paraformaldehyde for 15 min at room temperature. were labeled with PKH26 to ensure their traceability and
Thereafter, they were permeabilized with 0.1% Triton visualization. These cells were subsequently 3D bioprinted
X-100 for 10 min and blocked using 1% BSA in PBS for 1 h. to create PMCaTs. After a 5-day culture period, Jurkat cells,
an immortalized line of human T lymphocyte cells, were
For the identification of endothelial cell markers,
tissues were incubated overnight at 4°C with fluorescently labeled with CellBrite Green. These labeled Jurkat cells
conjugated antibodies targeting CD31 (at a 1:200 dilution were then thoroughly mixed with collagen hydrogel, and
in 1% BSA/PBS). Following three washes with PBS, these the PMCaTs were delicately embedded within this mixture.
tissues were set up with a mounting medium optimized for Once embedded, the collagen was allowed to polymerize,
fluorescence microscopy. Confocal imaging was executed securing the encapsulation of both the PMCaTs and Jurkat
using a Nikon confocal system, maintaining consistent cells. After the collagen polymerization, the entire system,
image acquisition settings across samples. To visualize the which comprised the PMCaTs surrounded by Jurkat
intricate 3D structure of the newly formed microvascular T cells, was visualized and imaged using a fluorescent
networks inside the tissues, Z-stacks were captured microscope to study the distribution and interaction of the
and then 3D reconstructed, adhering to our previously labeled cells.
documented protocol. 17 2.12. Modeling printed micro-cancer
tissue metastasis
2.9. Invasion assay
To examine the invasive properties of our PMCaTs, we SUM149 cells, NHLFs, and HUVECs were labeled with
utilized an invasion assay. The PMCaTs were embedded PKH26 and bioprinted into PMCaTs (as detailed in
section 2.5. Bioprinting process). Given that the half-

Volume 10 Issue 3 (2024) 561 doi: 10.36922/ijb.2911