Page 425 - IJB-10-2

International Journal of Bioprinting In vitro 3D pancreatic acinar unit

water solutions starting from 30/70 up to 100/0. finally mounted on glass coverslips using Fluoromount™
Before analysis, all the samples were coated with a thin Aqueous Mounting Medium (00-4958-02, Invitrogen)
platinum layer. for imaging. All samples were then imaged by confocal
microscopy (Eclipse Ti2, Nikon). The resulting images
2.7. Epithelization of the cavity were post-processed and analyzed with ImageJ software.
HPDE-KRAS and HPDE-WT cells were detached from the In particular, the fluorescence intensity (mean gray
culture flask, counted, and resuspended to a 10 μL volume values along the z-axis) corresponding to the E-cadherin
to obtain a 2.1 × 10 cells/mL concentration. Then, cells signal was analyzed in z-stack acquisitions and plotted to
6
were seeded into the cavity of cellularized MEW scaffold, compare the epithelial cell distributions within the cavity
where HFF1 were allowed to grow for 2 weeks. The co- of the MEW scaffold at different time points of culture
cultured constructs were placed in a 48-well plate. period. The fluorescence intensity values were normalized
The HPDE cells/HFF1 cell ratio was set at 1:3, according to the minimum value measured by the software for
to studies demonstrating the relevancy that ratios of 1:1 to each sample.
1:3 have in vivo. 42-44 Cells were co-cultured in DMEM/F-12
supplemented with 15% FBS (Gibco), 1% penicillin– 2.9. Interleukin-6 cytokines release
streptomycin (Gibco), and 2% L-glutamine (Gibco) since The concentration of cytokines was determined in cell
previous experiments had proven the efficacy of this culture supernatants collected, after 2, 3, 10, and 14 days from
medium composition in promoting cell viability. The HPDE cells seeding, from the wells containing the MEW
21
constructs were then fully covered with 600 μL of medium scaffolds co-cultured with HFF1, HFF1 + HPDE-KRAS,
after 60 min. The cellularized structures were maintained and HFF1 + HPDE-WT cells. IL-6 cytokines were
in a humidified CO incubator at 37°C and 5% CO . quantified with the IL-6 Human ELISA Kit (BMS213-2,
2 2 Invitrogen). The concentrations were calculated using the
2.8. Immunocytochemistry and confocal microscopy standard curve generated by plotting the absorbance values
The co-cultured MEW constructs were fixed with 4% of each standard sample on the ordinate and the human
paraformaldehyde (PFA; Sigma Aldrich) for 30 min IL-6 standard concentrations on the abscissa.
at room temperature, after 3, 7, 10, and 14 days of co-
culture. They were then rinsed with PBS twice and kept 2.10. Statistical analysis
at 4°C. Each sample was then permeabilized with 0.2% All bar graph data are presented as the mean ± standard
Triton X-100 in PBS for 10 min at room temperature and deviation (SD) from at least three independent experiments
successively washed 3 times for 5 minutes with PBS + 0.1% per condition or time point. Significance was measured
Tween 20 (PBST) at room temperature. Then, samples as indicated for each experiment, with two-way or one-
were blocked with 2% BSA in PBST for 60 min and rinsed way analysis of variance (ANOVA) followed by pairwise
three times with PBST. The samples were incubated comparison with Tukey’s multiple comparisons test using
with primary antibodies—alpha-smooth muscle actin GraphPad Prism 9.3.1; *p < 0.05, **p < 0.01, ***p < 0.001,
recombinant rabbit monoclonal antibody (1:200; 701457 ****p < 0.0001.
100 µg, Invitrogen) and E-cadherin monoclonal antibody
(HECD-1; 1:2000; 13-1700, Invitrogen)—in PBST + 1% 3. Results
BSA for 24 h at 4°C. Samples were then rinsed 3 times 3.1. Fabrication of acinar structures
with PBST at room temperature for 5 min. Then, the The effect of the process parameters on the resulted MEW
samples were incubated with secondary antibodies— scaffolds was evaluated qualitatively by observing the
Cyanine5-conjugated goat anti-mouse secondary print shape fidelity with respect to the CAD design at the
antibody (1:200; A10524, Invitrogen) and Alexa Fluor macroscopic and microscopic levels, and quantitatively by
TM
555-conjugated goat anti-rabbit secondary antibody measuring the diameter of fibers. The SEM images in Figure
(1:500; A27017, Invitrogen)—in PBST + 1% BSA for 2 2a show representative images of MEW scaffolds produced
h at room temperature. Samples were rinsed with PBST by varying the process parameters in the ranges reported
for 5 min at room temperature thrice. The cytoskeletons in Table 1. Specifically, the structures were obtained with:
of HFF1 and HPDE cells seeded in MEW scaffolds were 0.5 mm (i–iii) and 0.3 mm (iv) nozzle size; 20% (i, iv) and
TM
then stained with Alexa Fluor 488 Phalloidin (A12379, 40% (ii, iii) flow; 80 µm (i, iv) and 110 µm (ii, iii) infill line
Invitrogen) at 1:60 concentration in a solution of 1× PBS distance; 45° (i, ii) and 90° (iii, iv) infill rotate angle; 70
with 1% BSA. Samples were rinsed with PBS for 5 min at mm/s (i), 60 mm/s (ii, iii), and 40 mm/s (iv) speed; 7 mm
room temperature thrice, before the staining with DAPI (i), 6 mm (ii, iii), and 12 mm (iv) distance; 130°C (i), 100°C
(D1306, Invitrogen) in PBS (1:1000) for 5 min at room (ii, iii), and 90°C (iv) printing temperature; and 5 kV (i, iii,
temperature. They were then washed once with PBS, and iv) and 7 kV (ii) voltage.

Volume 10 Issue 2 (2024) 417 doi: 10.36922/ijb.1975