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International Journal of Bioprinting 3D-bioprinted macrophage inflammation model

from harvested cells using ISOLATE II RNA Mini Kit 6-diamidino-2-phenylindole; Sigma-Aldrich, Australia),
(Meridian Life Science, Australia) and quantified using and 0.8 U/mL TRITC-Ph (tetramethylrhodamine-
Thermo Fisher Scientific NanoDrop ONE (Australia). conjugated phalloidin; Sigma-Aldrich, Australia) in PBS
c
Complementary DNA (cDNA) for the isolated RNA was for 1 h at room temperature. The final constructs were
prepared using SuperScript III First-Strand Synthesis kit imaged using Nikon Eclipse Ti confocal microscope.
™
(Invitrogen) in SimpliAmp Thermal Cycler (Applied Excitation/emission wavelengths used for the secondary
Biosystems by Thermo Fisher Scientific, Australia). Real- antibody (for iNOS in green color), DAPI (for nucleus
time quantitative polymerase chain reaction (RT-qPCR) in blue color), and TRITC-Ph (for actin filaments in red
for M1 (IL-1β, IL-6, iNOS, and TNF-α) and M2a (CD206 color) were 488 nm/500–550 nm, 405 nm/417–477 nm,
and DC-SIGN) markers was performed with StepOnePlus and 561 nm/570–1000 nm, respectively.
™
Real-Time PCR System (Applied Biosystems) using SYBR
®
Green master mix (Invitrogen). The forward and reverse 2.8. Action of Ibu in the 3D-bioprinted
primers used for the RT-qPCR are listed in Table S1 inflammation model
(Supplementary File). The recorded cycle threshold (C ) THP-1 were 3D-bioprinted, differentiated to M0 (PMA,
t
values were used to calculate the fold change using the 25 ng/mL for 2 days and resting for 1 day), and polarized
Livak method (ΔΔCt method). The fold change (2 −ΔΔCt ) to M1 (LPS, 1000 ng/mL for 2 days). The concentrations
27
is the relative gene expression (2 −ΔCt ) in the 3D-bioprinted of 10 and 15 µg/mL Ibu (Sigma-Aldrich, Australia) were
28
constructs divided by the 2 −ΔCt of the 2D-cultured M0. selected for the study based on Ibu pharmacokinetics. The
M1-polarized constructs were incubated with 10 µg/mL
2.6. Optimization of LPS dose (Ibu1) and 15 µg/mL Ibu (Ibu2) in BM for 3 days, with M1-
Activation of M1 macrophages within the 3D-bioprinted polarized constructs without Ibu and M0-differentiated
constructs in the presence of increasing doses of Ec-LPS constructs as negative and positive control, respectively.
was investigated. Bioprinted THP-1 were differentiated to Test, negative control, and positive control constructs
M0 using the optimized PMA dose of 25 ng/mL for 2 days. (n = 3) were collected on days 1 and 3 for gene expression
After 1 day of rest, the culture media (1 mL/construct) were analysis (described in section 2.5). Relative gene expression
replaced with BM in the presence of increasing doses of Ec- was normalized to the relative gene expression of positive
LPS (0, 50, 100, 200, 500, 1000, 5000, and 10,000 ng/mL; control (M0) to calculate fold change. Phenotypic
n = 3) and incubated for 3 days. The conditioned media were characterization of M1 macrophages after 3 days of Ibu
collected and replenished with fresh media and respective treatment was performed via flow cytometry (n = 3) for
Ec-LPS doses at 1, 2, and 3 days. The release of M1-specific M0 markers (CD11b and CD33) and M1 marker (CD80).
cytokine (IL-6) in the conditioned media was quantified Conditioned media from each group were collected
using ELISA (Thermo Fisher Scientific, Australia) as per on days 1, 2, and 3 of culture, and ELISA was used to
the manufacturer’s instructions and normalized to the wet measure levels of secreted IL-6, IL-1β, TNF-α (Thermo
weight of each construct measured at day 3. Cells retrieved Fisher Scientific, Australia), and prostaglandin E2 (PGE ;
from collagenase-digested constructs were assessed for Cayman Chemical, USA). The cumulative quantity of
2
expression of the M1-specific surface marker CD80 by flow released protein was normalized with the wet weight of the
cytometry (section 2.4).
constructs. The effect of Ibu on the polarized 3D-bioprinted
2.7. iNOS expression M1 cells was also evaluated by measuring the secretion of
The expression of iNOS in M1-polarized cells within TNF-α and IL-1β using ELISA.
3D bioprinted constructs was visualized using 2.9. Statistical analysis
immunofluorescence. At day 3, non-polarized M0 All the data are presented as mean ± SD and were analyzed
constructs and M1-polarized (using optimal Ec-LPS dose using GraphPad Prism Version 8. One-way and two-
1000 ng/mL) constructs were fixed in 4% (w/v) PFA for way analyses of variance (ANOVA) with a Tukey post-
20 min and permeabilized using 0.2% (v/v) Triton X-100 hoc analysis were used to test for differences between
for 30 min. Afterward, the constructs were incubated in experimental and control groups. Statistical significance
blocking buffer (1% (w/v) bovine serum albumin and was set at p ≤ 0.05.
10% (v/v) normal goat serum in DPBS) for 2 h at room
temperature, then 200 ng/µL of anti-iNOS primary 3. Results and discussion
antibody (Abcam, UK) in blocking buffer for 24 h at
4°C. Thereafter, the constructs were incubated with 5 ng/ 3D bioprinting is an innovative additive manufacturing
µL secondary antibody Alexa Fluor 488-conjugated- technology that enables the construction of tissue-
®
anti mouse antibody (Abcam, UK), 5 µg/mL DAPI (4’, or organ-like structures through a layer-by-layer

Volume 10 Issue 2 (2024) 402 doi: 10.36922/ijb.2116