Page 260 - IJB-10-3

International Journal of Bioprinting Bioprinting organoids for toxicity testing

with each other in the DOs (Figure 2C). Compared was gradually detected in the DOs, while no substantial
with cultured planner cells before printing (BP), DOs increase was observed in empty microspheres (Figure 3A
showed hepatocyte maturation with upregulation of ALB and B), implying that the dynamics for the adsorption
and CYP3A4 and downregulation of the fetal gene AFP of microplastics into organoids are closely related to the
(Figure 2D). DOs exhibited transcriptional features of presence of organoids.
sinusoidal endothelial cells, with reduced expression of
CD31 and CD34, while elevated expression of LYVE1 To investigate whether PS-MPs diffuse in a targeted
and STAB2 (Figure 2D). Additionally, the activation manner into specific cell types, we labeled the cell
markers of HSC, such as ALCAM and COL1A1, were membranes of HEs, EPCs and LX-2 cells, respectively, and
downregulated in the DOs (Figure 2D). These results generated three kinds of DOs: HE-Green, EPC-Green, and
28
suggested that the hydrogel microspheres provided LX-2-Green. After an exposure for 8 h, the red signaling of
an appropriate microenvironment that facilitated the PS-MPs appeared within the range of EPCs but disappeared
assembly of multiple hiPSC-derived cells into organoids, from hepatocyte-like cells and LX-2 cells, indicating that
as well as mutual maturation with tissue-specific features. endothelial cells internalized PS-MPs more preferentially
Mechanically, RNA-seq analysis comparing DOs and BP than other cell types (Figure 3C). We reasoned that the
revealed upregulation of integrin signaling and calcium- mechanisms of cellular internalization differ among the
dependent cell–cell interactions as well as downregulation three cell types. 31,32 For example, PS-MPs larger than 1 μm
of cell proliferation and tight junction interactions (Figure could be internalized in human umbilical vein endothelial
2E). Presumably, these shifts in gene clusters contributed cells, but could rarely enter planar cultured hepatocytes.
33
31
to the morphogenesis and maturation of liver functions, We extended the exposure duration to 48 h and observed
including phase I/II metabolism, hormone response, MP signaling outside the region of endothelial cells,
VEGF production, and bile acid biosynthetic processes which indicates the diffusion of PS-MPs throughout the
(Figure 2F; Figure S2C in Supplementary File). organoids (Figure 3D).
To figure out the advantages of DOs in practical To study the influence of endocrine disruptors on
applications, we compared DOs with primary human PS-MPs accumulation in liver tissue, we added TBBPA
hepatocytes (PHH) which were considered the gold at environmental-relevant concentration (100 nM)
34
standard for evaluating in vitro hepatotoxicity. The to the fluorescence-labeled PS-MPs culture media to
results indicated that DOs exhibit higher transcriptional simulate PS-MPs/TBBPA co-exposure (henceforth
levels associated with sprouting angiogenesis and MPTA) (Figure 3D).We found that TBBPA significantly
response to thyroid hormone stimulus and endothelial aggravated the accumulation of PS-MPs in DOs at both
endocytosis (Figure 2G and H). These results indicated 8 and 48 h (Figure 3Eand F). This effect was likely due
that DOs were more representative than PHH in the to TBBPA altering the protein corona on the surface of
context of morphogenesis and response to toxicants. the microplastic, which as a result impacted its cellular
Taken together, we developed a biofabrication workflow internalization. These results probably explained
32
for the stable generation of liver Disse-like organoids
from hiPSC-derived monodispersed progenitor the synergistic toxicity of PS-MPs and TBBPA in a
12
cells. The biomimetic multicellular interaction, liver previous study and provoked a further exploration
morphogenesis, and diverse liver functions laid the of the synergistic toxicity of PS-MPs and TBBPA in
foundation for testing the multifaceted hepatotoxicity of multicellular organoids.
exogenous toxicants. 3.3. Co-exposure to PS-MPs and TBBPA showed

3.2. Entry and diffusion of the MPs and TBBPA in DOs limited toxicity on healthy donor-derived DOs
PS-MPs microbeads have been detected in human liver While the hepatic toxicity of microplastics and TBBPA
tissue samples ; however, the diffusion behaviors of MPs has garnered significant attention, there remains
5
in liver tissue are not yet clarified. Fluorescently labeled controversy regarding the effects on human liver health
microplastics allow for non-destructive visualization of at low-dose exposures. Herein, utilizing DOs derived
the distribution and accumulation of MPs in 3D structures from a healthy donor, we assessed the hepatic toxicity of
due to their minimal leakage of fluorescent groups in low-dose PS-MPs (600 ng/mL), TBBPA (100 nM), and
DPBS or culture media. 29,30 We observed and recorded their composite MPTA (600 ng/mL/100 nM) via the
the diffusion of fluorescently labeled PS-MPs microbeads evaluation of cellular toxicity, transcriptome vibration, and
in DOs-laden hydrogel microspheres. After incubating 1 hepatic functional impairment under short-term (3 days)
μm red fluorescence-labeled MPs for 1800 s, fluorescence exposure conditions.

Volume 10 Issue 3 (2024) 252 doi: 10.36922/ijb.1403