Page 424 - IJB-10-2

International Journal of Bioprinting In vitro 3D pancreatic acinar unit

(width) × 2.5 mm (thickness), while the central cavity irradiated with ultraviolet light for 1 h (30 min on each
was designed with a diameter of 1.4 mm. CAD models side) for sterilization purposes before cell seeding.
were discretized into triangles, and .stl files were thus Human fibroblasts were seeded in sterile MEW
generated. Then, the g-codes were obtained using the scaffolds placed in a 48-well plate. In particular, HFF1 were
Ultimaker Cura 4.8.0 software by setting the parameters seeded on MEW constructs by pipetting 40 μL of a 1.6 ×
needed to perform the slicing and later optimized using 10 cells/mL cell suspension on the top of the scaffolds. The
6
the Repetier-Host and NC Viewer v1.1.3 software. constructs were maintained at 37°C and 5% CO for 1 h
Specifically, the automated g-codes, generated by the .stl before 600 μL of medium was added to each well. 2
files slicing, were further edited to (i) prevent the needle
from passing over the scaffold between one layer and 2.5. Viability assay
the next, so as to avoid depositing undesired fibers; and The viability of human fibroblasts (HFF1) cultured in
(ii) implement an additional extrusion step at the initial MEW constructs was evaluated through the fluorimetric
phase of the printing process, in order to stabilize the resazurin reduction method (CellTiter-Blue, G8080,
jet. Then, NovaSpider v5 instrument (CIC nanoGUNE) Promega, Milano, Italy) 3, 7, 14, 21, and 28 days after
was employed to fabricate PCL (Mw ~ 43,000 Da; 19561- seeding. The assay was carried out according to the
500, Polysciences Inc., Warrington, USA) scaffolds manufacturers’ protocols. Briefly, culture medium was
by MEW. To achieve a printed structure with proper removed, and constructs were washed with 1× phosphate-
filament diameter, pore size, and shape fidelity, several buffered saline (PBS; Gibco). A solution of 16% CellTiter-
process parameters were tested, by varying their values Blue in complete cell culture medium was prepared and
in the ranges shown in Table 1. Ambient parameters added to the constructs. After an incubation period of
(i.e., chamber temperature and humidity) were set at 3–4 h at 37°C, 100 μL of the medium was pipetted into
27.5°C ± 3°C and 44% ± 2%, respectively. In detail, the different wells of a 96-well plate, and fluorescence was
humidity was monitored and controlled by an external measured using a plate reader (Synergy HTX Multi-Mode
humidifier (miniClima Humidity Control, miniClima Reader, BioTek) at 530 nm excitation wavelength and 590
Schönbauer GmbH, Wien, Austria) connected to the nm emission wavelength. All the obtained data were then
NovaSpider apparatus. exported and elaborated using GraphPad Prism 9.3.1.

2.3. Dimensional analyses 2.6. Stromal cells distribution within MEW scaffolds:
The effect of parameters on printed scaffold resolution confocal microscopy and scanning electron
was evaluated by scanning electron microscopy (SEM; microscopy analyses
Tescan Vega, Brno-Kohoutovice, Czech Republic) of MEW Fluorescence imaging was performed to monitor the
scaffolds. Before analysis, all the samples were coated with a distribution of stromal cells (HFF1) seeded within the
thin platinum layer. SEM images were then analyzed using 3D scaffolds. The constructs were washed once with 1×
ImageJ to quantify the average size of fibers. In detail, two PBS and fixed with 4% paraformaldehyde (PFA; Sigma
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scaffolds per condition were examined, and 40 values for Aldrich) for 30 min at room temperature, after 72 h
each scaffold were measured. of culture. They were then washed twice with 1× PBS,
permeabilized in 0.5% Triton X-100 (Sigma Aldrich) in
2.4. Seeding of stromal cells in MEW constructs 1× PBS for 10 min, and incubated with 1% bovine serum
At first, the MEW constructs were incubated in a solution albumin (BSA; Invitrogen, Carlsbad, CA, USA) for 30 min
containing 70% ethanol and 30% water overnight and then to enhance the quality of the staining. The cytoskeletons
of fibroblasts in MEW scaffolds were stained with Alexa
Table 1. Process parameters used to fabricate the MEW
TM
scaffolds Fluor 488 Phalloidin (A12379, Invitrogen) at 1:60
concentration in a solution of 1× PBS with 1% BSA.
Scaffold type i ii iii iv Nuclei were stained with DAPI reagent (4’,6-diamidino-
Nozzle size (mm) 0.5 0.5 0.5 0.3 2-phenylindole, Dihydrochloride; D1306, Invitrogen) at
Flow (%) 20 40 40 20 1:1000 concentration in 1× PBS solution. All samples were
Infill line distance (µm) 80 110 110 80 then imaged by confocal microscopy (Eclipse Ti2, Nikon,
Konan Minato-ku, Japan). The resulting images were post-
Infill rotate angle (°) 45 45 90 90 processed with ImageJ software.
Speed (mm/s) 70 60 60 40
Furthermore, SEM analyses were performed upon
Distance (mm) 7 6 6 12 dehydration of fixed fibroblasts within the scaffolds
Printing temperature (°C) 130 100 100 90 to observe both HFF1 cells and the MEW filaments.
Voltage (kV) 5 7 5 5 Samples were dehydrated by soaking them into ethanol/

Volume 10 Issue 2 (2024) 416 doi: 10.36922/ijb.1975