Page 447 - IJB-10-5

International Journal of Bioprinting Bioprinting for large-sized tissue delivery

the M14A transcriptome before and after bioprinting. and hepatic function maintenance with the potential for
Surprisingly, GSEA indicated no significantly altered gene minimally invasive applications.
terms after printing (Figure 5F). The cluster heatmap of
the whole transcriptome was minimally altered, suggesting 4. Conclusion
that the gene expression profile was maintained after the This study proposed a decoupled bioprinting strategy
bioprinting procedure (Figure 5G). Taken together, the to fabricate large-sized architectures with outstanding
cell viability test and RNA-seq analysis revealed the high deformation and shape recovery behavior using a novel
cell-bioprinting suitability of the decoupled bioprinting bioink featuring superelasticity and fatigue resistance. The
strategy using GP bioink and white light-induced printed architectures presented geometry-independent
photocrosslinking, even for sensitive gene-edited cells.
injection capacity and potential applications for minimally
3.5. Long-term culture and injection experiment of invasive tissue delivery. The development of GP represents
cell-laden bioprinted architectures a paradigm shift from mechanically reinforced material
The M14A-laden bioprinted architectures were cultured for development to biological performance. GelMA and
16 days. During culture, the architectures maintained good PEGDA of large M formed a reinforced crosslinking
w
structural stability with slight degradation and shrinkage network with improved mechanical properties,
of hydrogel filaments, suggesting the suitability for long- particularly superelasticity and fatigue resistance. We
term cell culture with degradation time comparable assume the unique mechanical properties were attributed
to tissue regeneration (Figure 6A). More importantly, to the incorporation of flexible chains during white
M14A cells embedded within GP hydrogel architectures light-induced photocrosslinking and the formation of
maintained high cell viability and proliferation. We macroporous structures. The superelastic GP hydrogel
observed the formation of cellular aggregates around expressed remarkable printability for 3D printing to
day 9, suggesting cell survival and growth (Figure 6B). obtain customized off-the-shelf architectures with distinct
Immunofluorescence staining on day 16 displayed positive features and tunable Poisson’s ratios. The large-sized
expression of hepatic specific marker MRP2, suggesting architectures can be compressed to less than 1% of their
that cells form tight junctions in aggregates (Figure 6C). original sizes and injected through nozzles comparable
Chemical staining was also performed to evaluate hepatic to 14G syringe needles. This injection capacity is bioink-
metabolic function. Glycogen PAS staining revealed that dependent and geometry-independent, offering greater
the cellular aggregates expressed high glycogen storage freedom for structural design and architecture scale.
capacity (Figure 6D). ICG uptake and release experiments The bioprinting of GP is compatible with cell-laden
reported good endocytosis and exocytosis behavior of hydrogel. Additionally, a genome-edited hepatic cell
M14A within the GP hydrogel, with successful ICG line M14A was bioprinted and long-term cultured as a
uptake within 30 min and complete release after 8 h paradigm. A cell-bioprinting suitability test was proposed
(Figure 6E). GP hydrogel proved to be a versatile candidate by combining live/dead staining with RNA-sequencing
for bioprinting and long-term culture with mechanically analysis to provide a full-scale visualization of the cellular
reinforced behavior and acceptable biocompatibility. alternations after printing. The decoupled bioprinting
However, we found that the cell proliferation in GP- strategy using GP hydrogel demonstrated high cell-
bioprinted architectures was significantly slower than bioprinting suitability, high cell viability, and no significant
the cells cultured in other biocompatible matrices (e.g., alterations in the transcriptomic profile. During the 16-day
alginate and collagen), highlighting the need for improving culture, GP hydrogel expressed good structural stability
material biofunctionality. and biocompatibility to support M14A cell survival and
function maintenance. The cell-laden architectures could
An injection test was performed on day 16. After injection
via 1.5 mm dispensing needles, the samples did not exhibit be successfully injected without structural fracture and
with minimal impact on the cells. This proof-of-concept
any filament fracture and maintained the integrity of their study provides an alternative approach for efficient tissue
geometric features (Figure S13, Supporting Information; delivery in minimally invasive treatments.
Video S6, Supporting Information). Additionally, M14A
cells displayed high viability compared to samples before Acknowledgments
injection, suggesting the little influence of the injection
procedure on cell viability (Figure 6F). Taken together, The authors acknowledge Qiaobing Xu from Tufts
cell-laden GP-bioprinted architectures could support long- University and Liqun Wang from Zhejiang University for
term cell culture with good structural stability, providing their advice on the reinforcement mechanism investigation
a biocompatible microenvironment for cell aggregation and assistance with manuscript writing.

Volume 10 Issue 5 (2024) 439 doi: 10.36922/ijb.3898