Page 461 - IJB-9-6

International Journal of Bioprinting 3D bioprinting of in vitro cartilage tissue model

visualization of cell viability in the center of the constructs. Sciences, S9378) solution in PBS (Gibco, 20012-019) to
Samples were washed twice with phosphate-buffered saline dehydrate. They were then submerged in a 50:50 ratio
(PBS; Gibco, 20012-019) and then incubated at 37°C for mixture of 30% sucrose solution and OCT mounting media
30 min in LIVE/DEAD cytotoxicity assay (Invitrogen, (VWR chemicals, 361603E) overnight at 4°C. Samples
L3224) using a concentration of 5 µL of calcein-AM and were then placed in cryomolds filled with OCT (VWR
20 µL of ethidium homodimer per 10 mL of PBS (Gibco, chemicals, 361603E), snap-frozen in liquid nitrogen, and
20012-019). After incubation, constructs were rinsed cryosectioned at 8 µm using a ThermoScientific cryotome
twice using PBS (Gibco, 20012-019) and imaged using an FSE for further immunohistochemistry processing.
Olympus DP80 microscope. Multiple z-stacks were taken
at magnifications of 10× and 20× using FITC (488 nm) 2.10. Hematoxylin and eosin staining
and TRITC (532 nm) filters. Semi-quantitative evaluation Routine hematoxylin and eosin (H&E) staining of
of cell viability was performed using Fiji Image-J software bioprinted constructs and 3D cell pellets sections
(1.53t version). In summary, red and green image channels were conducted at time points: days 0, 7, and 14 on the
were separated, and three random regions of interest were cryosections. Excess OCT was removed by submerging
selected to perform manual cell counting. Average cell the slides in 70% ethanol. Hematoxylin staining was
survival percentages for each time point were calculated performed for 10 min, and subsequently, Scott’s water was
and compared to 3D controls; dead and live cells were used to ensure nuclei bluing. Eosin staining was done for
counted at each specific time point and not used for 15 s, followed by washing in 70% ethanol.
comparison between time points due to the cell viability
not being cumulative. 2.11. Immunofluorescence labeling
Cryopreserved sample sections were rehydrated for 10 min
2.8. DNA quantification with PBTD (PBS + 1.1% DMSO + 0.1% Tween 20) and
DNA quantification was assessed using a Quant-iT subsequently fixed for 10 min using 10% neutral-buffered
PicoGreen dsDNA Assay Kit (Thermo Fisher, P7589). formalin (Sigma Aldrich, HT501128). Sections were then
Bioprinted constructs and 3D cell pellets were mixed with blocked for 1 h at room temperature using PBTD and 5%
pre-warmed (37°C) protease solution (10 mg/mL) (Sigma bovine serum albumin (BSA; Sigma Aldrich, A2153).
Aldrich, P5147-1G) and pipetted up and down until the Primary antibody incubation was performed overnight
PeptiInk or cell matrix was dissolved. This mixture was at 4°C, and the slides were kept in a humidity chamber.
then incubated at 37°C for 5 min. Sequentially, these Collagen type II primary antibody (Invitrogen, PA1-36059)
samples were mixed with 500 µL of 2× TE Buffer from was diluted at a ratio of 1:50; aggrecan primary antibody
the Quant-iT PicoGreen dsDNA Assay Kit (Thermo (Abcam, ab3778) was diluted at a ratio of 1:100; SOX-9
Fisher, P7589), and 1% Triton X in UltraPure™ DNase/ primary antibody (Abcam, ab185966) was diluted at a ratio
RNase-Free Distilled Water (Thermo Fisher, 10977-035). of 1:100 in PBTD and 5% BSA solution. All sections were
Mixtures were incubated at 37°C for 30 min and then washed in PBS for 5 min after primary antibody labeling
placed at -20°C. Samples were subjected to three freeze- was performed.
thaw cycles before the assay was performed. PicoGreen
dye was diluted (1:200) in 2× TE buffer. A total of 100 µL Secondary antibody incubation was performed at
from each sample was pipetted in a black/opaque 96-well room temperature. For collagen type II labeling, secondary
plate. An additional 100 µL of the PicoGreen dye solution antibody AlexaFluor-488 goat anti-rabbit (Invitrogen,
was then added and left for 5 min under constant mixing A11008) was incubated for 1 h at a ratio of 1:200 combined
at room temperature. Fluorescence was immediately with phalloidin (Invitrogen, A12381) at a ratio of 1:200
measured using a plate reader at excitation and emission in PBS. Aggrecan labeling was performed with the
wavelengths of 480 nm and 520 nm, respectively, for DNA AlexaFluor 594 goat anti-mouse secondary antibody
concentration calculation. Change in DNA concentration (Abcam, ab150116) diluted at a ratio of 1:200 for 3 h in
was calculated against concentration at day 0 (100%). PBS. SOX-9 labeling was performed with AlexaFluor 555
goat anti-rabbit (Invitrogen, A21428) incubated for 1 h
2.9. Histological processing and cryosectioning at a ratio of 1:200 in PBS. All sections were then washed
3D cell pellets and bioprinted constructs were washed with for 5 min in PBS, and nuclei staining was performed by
PBS (Gibco, 20012-019) for 5 min at different time points incubating the sections for 3 min at room temperature
(days 0, 7, and 14) and then fixed overnight at 4°C in a with DAPI (Thermo Fisher, 62248) diluted at a ratio of
10% neutral buffered formalin solution (Sigma Aldrich, 1:200 in PBS. Further section rinsing was done for 5 min
HT501128). After fixation, samples were washed for 5 min in PBS, before section mounting with fluoroshield (Sigma
in PBS and then left for 3 h in 30% sucrose (Sigma Life Aldrich, F6182).

Volume 9 Issue 6 (2023) 453 https://doi.org/10.36922/ijb.0899