Page 408 - IJB-10-2

International Journal of Bioprinting 3D-bioprinted macrophage inflammation model

bioprinting. Different concentrations of GelMA (2.5, of FDA-bound cells within the total cell population (both
5, 7.5, 10, 12.5, 15% (w/v)) containing 0.15% (w/v) FDA and PI-strained cells).
photoinitiator lithium phenyl (2,4,6-trimethyl benzoyl) TotalnumberofFDA boundlivecells green( )
phosphinate (LAP; Sigma-Aldrich, USA) were mixed Cellviability(%) Totall numberof cells with green andred signals 100
at 37°C, loaded in sterile 3D bioprinting cartridges and
incubated at 23°C for 20 min for thermal crosslinking.
25 G (inner diameter of 260 µm) sterile tapered 2.4. Optimization of macrophage (M0)
dispensing tips were connected to the cartridges and differentiation in 3D-bioprinted THP-1
loaded in a BIOX 3D bioprinter (CELLINK, Sweden) The 3D-bioprinted THP-1 constructs were treated with
to optimize 3D bioprinting parameters (printing different doses of PMA, and evaluation of macrophage
pressure and speed). The 3D-bioprinted constructs differentiation was carried out by quantifying the number
were photo-crosslinked using a custom-made 15 × 12 of cells expressing the known M0 marker, CD11b. 24-26
cm LED panel (wavelength 405 nm and total power The bioprinted and photo-crosslinked constructs
2
3
20 W) at 1 cm curing offset for 1 min. The constructs (10 × 10 × 0.8 mm ) were transferred to 12-well plates and
were immediately transferred to Dulbecco’s phosphate- equilibrated overnight in BM. A comparative 2D culture
buffered saline (DPBS) for imaging to calculate the for macrophage differentiation was prepared by seeding
5
printability (Pr) as described below. 5 × 10 THP-1 cells into the wells of 12-well culture plates
in 1 mL of BM. After equilibration, the 3D-bioprinted
The 3D-bioprinted constructs with 0–90° alignment constructs and the 2D controls were incubated with 0, 10,
were imaged using a stereomicroscope (Leica EZ4E), and 25, 50, 100, and 200 ng/mL PMA in BM for 2 days and
the square grids’ geometry (area A and perimeter L) was then equilibrated again in BM (no PMA) for another day
measured with ImageJ software. The printability (Pr) of (n = 3). The PMA-treated cells in 2D were harvested for
the constructs was quantified using Equation I. A Pr value flow cytometry by treating with TrypLE Express Enzyme
™
between 0.9 and 1.1 is considered to be an acceptable (Gibco , Australia) for 10 min at 37°C. The macrophages
®
printability range. 22 were retrieved from the 3D-bioprinted constructs by
completely digesting the GelMA constructs (10 × 10 ×
(I) 0.8 mm ) 200 µL of 1 mg/mL (285 U/mL) collagenase
3
(Thermo Fisher Scientific, Australia) for 10 min at 37°C.
Based on the printability evaluation, 7.5% and 10% The optimization of collagenase digestion parameters
(w/v) GelMA was used for THP-1 3D bioprinting. THP- (concentration/treatment time) is described in Figure S1
6
1 (2 × 10 ) were pelleted from the suspension culture by (Supplementary File).
centrifugation at 200 × g for 3 min, washed twice with The harvested cells were washed with DPBS and
DPBS, and redispersed in 7.5% and 10% (w/v) GelMA stained with Fixable Viability Stain 510 (BD Biosciences,
containing 0.15% (w/v) LAP. The prepared bioinks USA) for 20 min at room temperature (23°C). Cells were
were loaded into a BIOX 3D bioprinter (CELLINK, incubated with Human BD Fc Block (BD Biosciences,
™
Sweden) and bioprinted using the optimized printing USA) for 10 min and stained with a panel of surface
conditions (Figure 1). markers. CD90, CD11b, and CD80 were used as negative
marker, macrophage (M0 and M1) marker, and M1
2.3. Assessment of 3D-bioprinted THP-1 viability marker, respectively. Data were acquired using a BD
through live/dead staining LSRFortessa Cell Analyzer supported by BD FACS Diva
™
The viability of THP-1 immediately (day 0) and 1 and 3 software. Cell populations were identified on FlowJo (Tree
days post-printing was assessed using fluorescent live/dead Star, Ashland, OR).
staining (n = 3) as previously reported. The 3D-bioprinted
23
constructs were stained using 5 µg/mL fluorescein diacetate 2.5. Role of 3D microenvironment in
(FDA; Sigma-Aldrich, Australia) and 2 μg/mL propidium macrophage polarization
iodide (PI; Sigma-Aldrich, Australia) for 30 min, then The polarization of M0 in 3D-bioprinted constructs in the
imaged using Nikon Eclipse Ti confocal microscope for absence of stimulatory factors such as LPS was assessed by
FDA-bound live (green) THP-1 (excitation [Exc] 488 nm/ investigating gene expression. Bioprinted THP-1 within
emission [Ems] 500–550 nm), and PI-bound dead (red) the 3D constructs (n = 3) were differentiated to M0 using
THP-1 (Exc 561 nm / Ems 570–1000 nm). The number of PMA and equilibrated in BM. Cells were harvested at 6 h,
green and red THP-1 cells was determined using ImageJ and 1, 2, and 4 days after collagenase digestion and stored
software, and the viability was calculated as the percentage at -80°C for future assay. Total RNA from was isolated

Volume 10 Issue 2 (2024) 400 doi: 10.36922/ijb.2116