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International Journal of Bioprinting 3D-bioprinted macrophage inflammation model

assembly of cells, growth factors, and/or other bioactive proliferating cells formed aggregates, and individual
components. This groundbreaking technology has cells could not be identified.
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found application in cancer modeling and served as
a robust and reproducible approach for investigating 3.2. Optimization of macrophage (M0)
novel anticancer drugs. 9,30,31 However, the exploration of differentiation in 3D-bioprinted THP-1
3D-bioprinted macrophages for simulating inflammatory PMA is commonly used to differentiate THP-1 cells into
cells that mimic human monocyte-derived macrophages,
conditions and responses is still in its infancy. with characteristics of decreased proliferation and
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Recent research has demonstrated the practicality of increased cell attachment, phagocytosis, and expression
3D-bioprinted structures consisting of polarized (M2) of multiple macrophage markers and cytokines. 25,34-36
macrophages as a fresh approach for disease modeling Since small differences in culture conditions, including
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and various applications in tissue engineering. However, PMA concentration, can affect the differentiation and
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there is a significant scarcity of data when it comes polarization of THP-1 cells, 37-39 it is essential to determine
to modeling inflammation using pro-inflammatory the most appropriate conditions for differentiation when
(M1) macrophages. Recent advancements include the using these cells as a model.
development of a bioprinted lung tissue model aimed at
studying inflammation and assessing the effectiveness of The PMA dose response toward the M0 differentiation
viral inhibitors. The researchers integrated THP-1 cells of THP-1 cells cultured in a 2D environment has been
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38-40
into a multicellular model and measured the release of extensively investigated. However, the PMA dose
pro-inflammatory cytokines, specifically IL-1β and IL- response of THP-1 cells in a 3D environment is not well
explored, as most of the research has focused on using a
8, following LPS stimulation. Nevertheless, the study fixed PMA concentration between 100 and 300 nM. 41-46 A
falls short of providing a comprehensive exploration previous research—the only study on the M1 polarization
of macrophage polarization and does not address the of 3D-bioprinted THP-1 cells—also used a fixed
model’s capability to respond to external stimuli, such as concentration of PMA (200 ng/mL= 324.2 nM).
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anti-inflammatory drugs. The current study developed
a 3D-bioprinted macrophage model and assessed its Here, M0 differentiation of THP-1 cells in 2D
inflammatory response to bacterial endotoxin (LPS). culture and 3D construct was quantified using different
Additionally, the effectiveness of an anti-inflammatory concentrations of PMA using flow cytometry (Figure 1d).
drug (Ibu) in inhibiting the inflammatory response of the The expression level of M0 marker, CD11b, and M1
3D-bioprinted macrophages was investigated. marker and CD80 was determined from live single cells
retrieved from 3D or 2D culture (Figure 1e and f). All
3.1. Viability of 3D-bioprinted THP-1 PMA concentrations tested stimulated similar levels
The printing pressure and speed for different of CD11b expression in 2D culture. The 3D-bioprinted
concentrations of GelMA are presented in Figure 1b. cells also responded to all PMA concentrations tested
Macrophages are tissue resident cells, and most with similar CD11b expression to that observed in
inflammatory conditions impact connective tissues 2D culture (Figure 1e). Maeß et al. showed that PMA
and resident organs, and as such, it is relevant that concentrations higher than 25 ng/mL resulted in higher
macrophages are tested for their ability to model expression of M1-related genes (IL-1β, TNF-α, and IL-8)
inflammatory responses in an environment that in 2D-cultured THP-1 cells and might mask the effect of
resembles the native extracellular matrix, as is the case subsequent stimuli. 38,39 Therefore, the influence of PMA
with GelMA. Based on the optimal printability of the on M1 polarization of 3D-bioprinted THP-1 cells was
constructs (Pr = 0.9 to 1.1; Figure 1b), 7.5% and 10% tested by quantifying the expression of the M1 surface
(w/v) GelMA was used for cell printing. The viability of marker CD80 (Figure 1f). The exposure of THP-1 cells
THP-1 cells was quantified using live/dead imaging, as with the higher dose of PMA resulted in increased
shown in Figure 1a and c. Immediately after bioprinting expression of CD80, and as such, 25 ng/mL PMA was
with 7.5% (w/v) GelMA, THP-1 viability remained used for M0 differentiation of 3D-bioprinted THP-1 cells.
comparable with the viability of cells before mixing 3.3. Role of 3D microenvironment in macrophage
with the GelMA bioink (82 ± 5%). However, the higher polarization
concentration of bioink resulted in increased printing Macrophages (M0) are equipped with a wide range of
pressure and a significant decrease in THP-1 viability, surface receptors that detect their environment and undergo
as observed in the bioprinting of other cell types. 23,33 polarization. Studies have shown that different properties
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Cell viability at day 3 could not be quantified as the of biomaterials and hydrogels, such as surface chemistry,
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Volume 10 Issue 2 (2024) 403 doi: 10.36922/ijb.2116