Page 419 - IJB-10-6

International Journal of Bioprinting 3D-bioprinted respiratory disease model

Briefly, 24-well plates were prepared for printing by bronchioles. These ventilation conditions induce both
adding 1 mL 0.1% polyethyleneimine (PEI; molecular shear and hydrostatic forces on the cells, acting as a relevant
weight [Mw]: 60,000, 50% [w/w] aqueous solution, biomechanical stimulus. It has been recently illustrated that
J61270.22; Thermo Scientific, USA) and incubating the incubation of constructs in such dynamic conditions
overnight in an incubator at 37°C. The 0.1% PEI solution affects cell proliferation, with variations observed during
was then removed and replaced with 0.5 mL 50 mM light, normal, and heavy breathing conditions, compared
CaCl with 0.1% PEI as the crosslinking solution. Using to static culture conditions. 34
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a GeSiM mbH BioScaffolder 3.2 (GeSiM, Germany)
outfitted with a 27-gauge needle (inner diameter: 200 2.7. Printability
µm), the synthesized bioink was pneumatically extruded Printability was assessed by printing the bottom two
layers of the cylindrical scaffold design and characterizing
at 37°C into the prepared 24-well plates. Constructs them by measuring the strand diameter, maximum
remained submersed in the crosslinking solution for 10 and minimum diameter, and pore size, as well as an
min before the crosslinking solution was replaced with assessment of scaffold repeatability using ImageJ software
3D culture media (50% HPF media, 50% HBEpC media, (n = 72). The printed scaffolds were submerged in the
20 mM CaCl ). Notably, the use of PEI in bioprinting crosslinking solution for 10 min and then imaged on a
2
has toxic effects on the incorporated cells, and high BioTek Lionheart LX automated fluorescence microscope
concentrations of CaCl can also negatively impact at 4× magnification. Printability analysis was carried out
2
cell viability. Therefore, scaffolds were printed into the for scaffolds both with and without nanoparticles and
crosslinking solution (50 mM CaCl + 0.1% PEI) and with varying printing parameters, including temperature,
2
immersed for only 10 min, allowing for appropriate pressure, and print head speed, for each test, as these
crosslinking and limited toxicity. The inclusion of the factors have been reported to affect printability. 46,47 To
lower concentration of 20 mM CaCl in the culture media minimize uncertainties in the assessment, printing
2
allowed for further crosslinking throughout the culture parameters were selected to ensure consistent and
period, thus maintaining the structural integrity without repeatable strand printing while creating the designed
negatively impacting cell viability. scaffolds, instead of primarily basing selection on meeting
Each construct was bioprinted with approximately 1.3 a specific strand dimension.
× 10 HPFs and 1 × 10 THP-1s for subsequent biological 2.8. Degradation
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studies. Specifically, the constructs underwent a 7-day Degradation studies were carried out using cell-free
standard submerged culture period post-printing and then scaffolds in both static and dynamic culture conditions to
were seeded, with each construct receiving 5 × 10 HBEpCs determine the material loss over a 28-day culture period.
4
on its pool surface. At 24 h post-seeding, air-liquid interface Briefly, 3D-printed constructs were transferred to 3D
(ALI) conditions were created through the reduction of culture media (50% HPF media, 50% HBEpC media, 20
media levels below that of the pool surface, exposing the mM CaCl ) after being immersed in the 50 mM CaCl
2
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epithelial cell-seeded surface. The start of ALI culture was crosslinker for 10 min. The plates were then moved into
considered day 1 for all biological experiments. It should a standard or ventilated incubator for culturing at 37°C.
be noted that constructs used in studies on printability, In the dynamic condition, the incubation chamber was
degradation, and mechanical properties (as discussed ventilated in a cycle that mimicked normal breathing
below) did not incorporate cells for simplicity and cost- conditions with biomechanical stimuli, including airflow
effectiveness. As suggested by previous studies, 33,34,45 the and pressure changes, as previously described. At
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involvement of cells has a limited effect on the construct’s the timepoints of 1, 7, 14, and 28 days, scaffolds were
printability, degradation, and mechanical properties over removed (n = 12), freeze-dried for 24 h, and weighed for
the timeframes of interest. determination of mass loss over time, with day 1 used as
the control.
2.6. Incubation
Incubation in dynamic conditions was carried out in a 2.9. Compressive testing on scaffold materials
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customized incubator, as previously described. Briefly, Cell-free bulk material specimens were created and used
the bioreactor induces cyclic pressure changes in the for compression testing to determine the bulk material
incubation chamber, mimicking both the pressure and compressive modulus. Briefly, the prepared solution was
air-flow changes that occur in the lung. Instead of directly poured into cylindrical molds before being immersed in
imposing mechanical stretch on the 3D-bioprinted 100 mM CaCl . The molds remained immersed for 48
2
constructs, the incubator mimics the ventilation h at 25°C to ensure complete crosslinking through the
conditions of the breathing cycle as seen in the bronchi/ cross-section of the samples. The higher concentration of

Volume 10 Issue 6 (2024) 411 doi: 10.36922/ijb.3895