Page 444 - IJB-10-5

International Journal of Bioprinting Bioprinting for large-sized tissue delivery

S function remodeling. Therefore, providing a comprehensive
Injection capacity = inject × 100% (IV) and scientific description of cell-bioprinting suitability is
S architecture indispensable.

Interestingly, all large-sized architectures exhibited In this study, we evaluated the cell-bioprinting
similar injection capacity, regardless of geometrical features suitability of GP hydrogel using two cell types: HepaRG cell
and Poisson’s ratios (Table S4, Supporting Information; lines and genome-edited hepatocytes based on HepaRG.
Video S3, Supporting Information). These architectures HepaRG is a human hepatoma cell line with bipotent
could be consistently compressed to 0.79% of the original hepatic progenitor-like characteristics and is readily used in
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size, injected through needles with a minimum inner artificial liver construction and drug testing applications.
diameter of 1.5 mm (comparable to a 14G needle), and The influence of light exposure time on HepaRG viability
subsequently unfolded and restored to the original shape was tested to determine the optimal window for cell-laden
while maintaining structural integrity (Figure 4C; Video bioprinting. Our findings indicate that 5 and 10 min of
S4, Supporting Information). When the needle diameter exposure did not significantly influence cell viability in the
decreased to 1 mm, all the samples were damaged, with absence of added culture medium. However, long-term
fractured filaments (Figure S9, Supporting Information). exposure (15 and 20 min) induced massive cell death,
These results revealed that the deformation and shape likely due to liquid evaporation and heat radiation from
recovery behavior of large-scale architectures was versatile, white light exposure (Figure 5A). A compressive test was
depending on the inherent mechanical properties of GP, then conducted on GP that was crosslinked for 10 min.
and compatible with different structural designs for The elastic modulus was determined to be 23.2 ± 1.6 kPa
injection procedures. (Figure S11, Supporting Information), i.e., within the
scope of human tissues and comparable to the natural
After assessing the injection capacity of large-sized 38,39
architectures, a simulation experiment was conducted liver. Therefore, 10 min light exposure time was optimal
for subsequent cell-laden bioprinting studies.
for in vivo injection. The sinusoidal mesh samples were
injected via a dispensable syringe with an inner diameter While cell viability is commonly used to assess the
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of 2 mm onto the surface of a pork liver (Figure S10, influence of the printing procedure, we consider it a
Supporting Information). The printed architecture rough analysis because cell injury may alter cell phenotype,
could be continuously injected and effectively spread on leading to unwanted variations in subsequent cell self-
the surface of liver tissue, suggesting the potential for organization and morphogenesis. Thus, we proposed
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minimally invasive tissue delivery applications (Video a paradigm for assessing cell-bioprinting suitability by
S5, Supporting Information). Furthermore, biomimetic combining cell viability and transcriptome sequencing
models of analogous liver lobules (radiation sinusoid (RNA-sequencing analysis) to provide a full-scale
model and hexagonal lobule model) were designed. The evaluation of cellular alternations after printing.
corresponding architectures were successfully printed We established a new genome-edited hepatic cell line
using a multi-nozzle extrusion printer with temperature- based on HepaRG to assess the cell-bioprinting suitability
control modules (Figure 4D). Taken together, we present of GP-bioprinted hydrogel. By transfecting two genes
a versatile bioprinting strategy for fabricating injectable (FoxM1 and HNF4A) using Sleeping Beauty transposon
large-sized architectures using GP hydrogel. Our findings into HepaRG (Figure 5B), we obtained fluorescent-
highlight that the excellent injection capacity was
dependent on the mechanical behavior of GP hydrogel labeled HepaRG-M14A cells (henceforth referred to as
and was compatible with various model designs featuring M14A) that exhibit the typical polygonal morphology of
tunable Poisson’s ratios. After injection, the samples were hepatocytes (Figure 5C). Additionally, these cells could
able to recover their original geometries. Hence, the spontaneously differentiate into hepatocytes without
versatility of GP hydrogel for bioprinting applications can a HepaRG induction medium. Immunofluorescence
be exploited to create intricate architectures with multiple staining revealed that M14A expressed high levels of
materials and biomimetic designs. hepatic marker proteins, i.e., HNF4A and ALB (Figure
5D). Moreover, M14A expressed more glycogen storage
3.4. Cell-laden GP hydrogel supports high and better ICG uptake capacity compared to HepaRG
cell-bioprinting suitability (Figure S12A, Supporting Information). RNA-sequencing
Cells are living components in the bioink and are critically analysis displayed a significantly different transcriptome
important for in vitro tissue/organ reconstruction. After for M14A compared to HepaRG (Figure S12B, Supporting
printing, a high cell survival rate and unaffected phenotype Information). Multiple gene sets related to biosynthesis,
are the prerequisites for subsequent cell assembly and metabolism, viral genome replication, and virus receptor

Volume 10 Issue 5 (2024) 436 doi: 10.36922/ijb.3898