Page 258 - IJB-10-3

International Journal of Bioprinting Bioprinting organoids for toxicity testing

set enrichment analysis (GSEA) was conducted using the embedding conditions (Figure S1I in Supplementary File).
local version of the GSEA tool (http://www.broadinstitute. Beyond four passages, the doubling time became prolonged
org/gsea/index.jsp). A protein–protein interactions (PPI) (Figure S1K in Supplementary File); thus, EPC-P4 cells
network of representative GSEA GO terms from the whole were used for subsequent organoid biofabrication.
clusters was constructed with Cytoscape EnrichmentMap.
The Pearson correlation heatmap, volcanic map, and gene 3.1.2. Electro-assisted bioprinting of hiPSC-derived DOs
heatmap were calculated and graphed using R (version While 3D bioprinting has been integrated with iPSCs
3.0.3) ggplot2 and pheatmap packages. The chord plot to 3D-print biomimetic tissues, it is crucial to select
was graphed with R GOplot v1.0.2. R (version 3.0.3). The an appropriate printing method based on the research
parallel set was graphed using the Origin 2021 software. objectives due to the distinct features associated with
20
All original sequence datasets were submitted to the GEO each technique. Extrusion-based bioprinting enables
database under the accession number GSE212099. the fabrication of hierarchical biomimetic models by
controlling spatial positioning of cell-laden bioinks.
2.11. Statistical analysis However, the high shear stresses involved in the printing
All values are expressed as the means ± standard deviations process can impair the cellular viability of iPSCs, which
(SD) and were analyzed using Prism software (GraphPad are sensitive to mechanical damage. 21,22 While the current
Inc.). Statistical analysis was performed using Student’s vat polymerization-based bioprinting technologies have
t-test or one-way analysis of variance (ANOVA). achieved superior bioprinting resolution and accuracy,
there is still a shortage of cell-compatible materials that
3. Results and discussion can be used with these technologies.The thermal inkjet
bioprinting technology is only applicable to a narrower
3.1. Fabrication of hiPSC-derived multicellular DOs range of biomaterials characterized by low viscosity. The
23
We sought to tackle the challenges of large-scale electro-assisted inkjet printing of hydrogel microspheres
biofabrication of biomimetic DOs from two main aspects: demonstrates compatibility with a wide range of
(i) obtaining sufficient hiPSC-derived progenitor cells that biomaterials, high printing throughput, and exceptional
facilitate the assembly of 3D structures and co-culturing structural stability. 24,25 Our previous study demonstrated
them with multiple cell types; and (ii) developing bioink that alginate-based microspheres generated by electro-
and biofabrication methods considering the survival and assisted inkjet printing maintained high cell viability of
subsequent multilineage maturation of hiPSC-derived cells. mesenchymal stem cells and hepatic stem cells in the

3.1.1. Specification of hiPSCs into progenitor cells printing process, and facilitated uniform cell aggregation
We differentiated hiPSCs derived from healthy donors and differentiation. 18,26 Thus, utilizing electro-assisted inkjet
(hiPSC-N) into HEs and EPCs, which retained the printing technology and a blend of alginate and laminin,
ability to self-assemble into liver organoids and showed we were able to generate a large amount of reproducible
synergistic development toward maturation. To obtain cell-laden microspheres measuring approximately 500
19
sufficient cells for biofabrication, HEs were sub-cultured μm in diameter (Figure 2A; Figure S2A in Supplementary
after differentiation into FOXA2+/HNF4A+ HEs (counted File). After 5 days of culture, the cell viability was almost
as passage 0; HE-P0) (Figure S1A in Supplementary File). fully recovered (Figure 2A). The three types of cells (HE/
Within the first three passages, HE-P3 positively expressed EPC/human HSC cell line LX-2) self-organized into
cell identity markers (Figure S1B and S1D in Supplementary multicellular organoids (DOs) with averaged diameters
File) and maintained their capacity for differentiating into of 35 ± 13 μm, the diameter of 90% of the organoids
hepatoblasts (HBs) and hepatocyte-like cells (HCs) with is within the range of 25–45 μm, and no necrosis was
the positive expression of ALB/AFP expression (Figure S1C observed within the center of the core (Figure 2A;
and S1F in Supplementary File). Beyond three passages, Figure S2B in Supplementary File). These results suggested
above markers exhibited decreased expression (Figure S1D that electro-assisted inkjet printing was suitable for printing
in Supplementary File); therefore, HE-P3 was used for monodispersed hiPSC-derived cells that were sensitive to
27
organoid biofabrication. Besides, hiPSCs-N differentiated mechanical disturbance.
EPCs (EPC-P0) (Figure S1G in Supplementary File) These DOs positively expressed hepatocyte typical
underwent planar expansion and further matured into markers (ALB+/CYP3A4+), endothelial markers
endothelial-like cells with upregulated expression of (CD31), and HSC markers (vimentin, VIM) (Figure 2B).
endothelial markers (Figure S1J in Supplementary File). We fabricated DOs-laden microspheres labeled with
A tube formation assay demonstrated EPC-P4 cells’ EPC-RFP, LX-2-EGFP, and nuclear staining. The results
capacity for in vitro angiogenesis on the Matrigel layer and indicated that the three types of cells closely interacted

Volume 10 Issue 3 (2024) 250 doi: 10.36922/ijb.1403