Page 483 - IJB-10-3

International Journal of Bioprinting Stretchable scaffold for modeling fibrosis

also FGM-3. After 24 h of cell culture, the medium was (Promega, USA) were performed after 7 and 14 days of
replaced with 100 µL of non-fluorescent resazurin solution culture. CellTiter-Blue solution (300 µL) was incubated
composed of CellTiter-Blue Cell Viability Assay reagent for 6 h with cellularized GelMA and PCL/GelMA samples,
(Promega, USA), diluted to 1:10 in the new FGM-3. Cell while CytoTox-ONE assay was performed on culture
viability was evaluated after a 4 h incubation in terms of medium from both samples following manufacturer
fluorescence intensity measured by a microplate reader instructions (see section 2.10.).
(BioTek Instruments, USA) at excitation (ex) and emission PCL/GelMA samples (with the three different GelMA
(em) wavelengths of 560 and 590 nm, respectively. concentrations) were cellularized with HCFs and cultured
Six measurements were performed for each hydrogel for 14 days in static conditions to assess the effect of
concentration to calculate the mean values and standard GelMA concentration on cell viability, spreading, and
deviations compared to control samples. cytoskeleton morphology. After selecting the optimum
2.11. Long-term cell viability and cytotoxicity tests GelMA concentration for cell spreading and cytoskeleton
on bioartificial stretchable scaffolds conformation, in vitro cell tests were repeated under
PolyDOPA-PCL scaffolds with two layers (6 × 4.5 mm , mechanical stimulation to assess its influence on HCF
2
considering the gauge length used in tensile tests) were activation into myofibroblasts. PCL/GelMA samples were
sterilized by incubation in 70% ethanol solution for 30 cultured for 7 days in static conditions (based on resultant
min and subsequently dried in a biological hood before cell morphologies within GelMA hydrogels in static
cell tests. conditions) and mechanical stimulation was subsequently
applied to the scaffolds for 7 additional days. The
Sterile GelMA_5, GelMA_7, and GelMA_10 solutions MechanoCulture T6 bioreactor (CellScale, Canada) was
were prepared as follows: (i) GelMA and LAP powders employed to cyclically stretch the PCL/GelMA samples
were UV-sterilized for 30 min in a biological hood (MSC- (4.5 × 1.5 unit cells) in the x–y plane (corresponding to
Advantage Class II Biological Safety Cabinet, Thermo 18 × 4.5 mm ) with a gauge length of 1.5 unit cells along
2
Fisher Scientific); (ii) GelMA powders were mixed with the x-axis (corresponding to 6 mm) and a four-layer
sterile FGM-3 and incubated at 50 °C under stirring for 2 thickness. The samples were subjected to cyclic mechanical
h until complete GelMA dissolution; (iii) LAP powder was deformations up to 10% maximum deformation (1 mm)
added to each GelMA solution in the sterile hood and the at 1 Hz frequency and incubated in FGM-3 at 37 °C. The
system was stirred at 50 °C for 15 min in dark conditions. experimental setup is displayed in Figure S6 and Video
HCFs were detached from the Petri dishes using S1, Supporting Information.
trypsin-EDTA and centrifuged to obtain cell pellets with 2.12. Immunofluorescence
defined amounts of cells (1,000,000 cells/mL), which were Cellularized PCL/GelMA scaffolds were fixed in 4%
then added to the GelMA_5, GelMA_7, and GelMA_10 w/v paraformaldehyde (Alfa Aesar, USA) in PBS for
solutions containing LAP at 37 °C. Cells were encapsulated 15 min and subsequently washed with PBS. The cells
by mixing GelMA solutions with cell pellets. Cellularized were permeabilized with Triton X-100 (Sigma-Aldrich,
GelMA solutions (30 µL) were deposed into polyDOPA- USA) 0.5% in PBS for 10 min. The samples were then
PCL scaffold pores and on 48-well plates as the control and blocked with 2% bovine serum albumin (BSA, Sigma-
cured under UV irradiation at specific times based on the Aldrich, USA) in PBS for 30 min, followed by either (i)
hydrogels’ rheological properties (30 s for GelMA_5 and staining with rhodamine-phalloidin (Life Technologies,
GelMA_7; 45 s for GelMA_10). After curing, viability and USA) or (ii) incubation with primary antibodies (i.e.,
cytotoxicity tests were performed. anti-actin smooth muscle [α-SMA; A7607, Sigma-
Live/Dead Cell Viability assay (Life Technologies, USA) Aldrich, US], anti-fibronectin [F3648, Sigma-Aldrich,
was performed after 1, 7, and 14 days. Briefly, cellularized USA], anti-collagen I [2456, Sigma-Aldrich, USA], and
GelMA hydrogels cultured in 48-well plates were treated anti-collagen III [SAB4500367, Sigma-Aldrich, USA]),
for 30 min with Live/Dead reagent (Life Technologies, and secondary antibodies (i.e., anti-mouse Alexa Fluor
USA), composed of 2 µM calcein acetoxymethyl (AM) and 555 [Life Technologies, USA] or Alexa Fluor 488 [Life
Technologies, USA]) diluted in 2% w/v BSA in PBS. Nuclei
4 µM ethidium homodimer-1 diluted in PBS. Samples were were counterstained with 4ʹ,6-diamidino-2-phenylindole
then imaged with a Nikon Ti2-E fluorescence microscope (DAPI; Sigma-Aldrich, USA). Samples were imaged
(Nikon Instruments, Japan).
using the Nikon Ti2-E fluorescence microscope (Nikon
Moreover, CellTiter-Blue Cell Viability Assay and Instruments, Japan). Immunofluorescence experiments
CytoTox-ONE Homogeneous Membrane Integrity Assay were performed in biological triplicates.

Volume 10 Issue 3 (2024) 475 doi: 10.36922/ijb.2247