Page 433 - IJB-10-2

International Journal of Bioprinting In vitro 3D pancreatic acinar unit

be obtained by incorporating the method of layer shifting cancer cells. The ability of this model in reproducing the
76
in the programmed toolpath, using planar collectors. 66,67 inflammatory cascade occurring in pancreatic cancer is
However, these scaffolds were poorly interconnected as further confirmed by numerous studies showing notable
fibers tend to adhere to each other. In contrast, we showed differences in serum IL-6 levels between PDAC patients
that the printing of complex biomimetic structures have and healthy individuals. 77-83
high shape fidelity and interconnected porosity, which are However, the differences in IL-6 levels of fibroblasts
key features for biological studies as confirmed by adhesion co-cultured with either healthy epithelial cells or epithelial
and proliferation assays using stromal cells (HFF1) that cells overexpressing KRAS were statistically significant only
remained viable and active for at least 4 weeks in MEW at early stages of the experiment, suggesting that the IL-6-
constructs (Figure 3). These results are supported by other mediated inflammation occurred mainly during the first
studies in literature, showing the culture of cells on PCL few hours (up to 72 h). At later phases of the experiment,
scaffolds obtained by MEW for several weeks. 61,68 Therefore, 84
our results confirm the ability of these biomimetic scaffolds in inflammation might be mediated by other proteins.
promoting cell growth and tissue formation, in line with the The ability of the human MEW model in reproducing
numerous studies reporting the large use of PCL in additive the natural compartmentalization typical of the exocrine
manufacturing approaches for biomedical applications. 37-40 pancreatic microenvironment was demonstrated, as the
The model developed in this study was able to maintain epithelial cells were localized within the cavity while
long-term culture of human fibroblasts, which adhered to fibroblasts colonized the 3D structure. Indeed, HPDE-
different fibers to create bridges across the pores and grew KRAS cells colonized the acino-like structure and remained
with the support of polymeric grid, imitating the natural collimated in the cavity up to 10 days of co-culture period
process. The surface of MEW scaffolds was almost covered and then started to migrate within the scaffold and on
by a thin layer of stromal matrix after 28 days in culture the scaffold upper surface (Figure 5c and d, and Figure
(Figure 4). 6; and Videos S1–S3 in Supplementary File). Therefore,
the optimal protocol for co-culture implementation
Moreover, we detected the presence of granular within the MEW structure was set: 14 days of fibroblasts
corpuscles on fibers of MEW models, at 21 days and 28 days culture alone plus 10 days of fibroblasts and epithelial
after HFF1 seeding. Evidence in literature seems to confirm cells co-culture. Indeed, these time points permit the
our hypothesis that correlates the presence of such corpuscles creation of a cellularized MEW model that mimics the
with the deposition of ECM by fibroblasts. 69,70 However, native compartmentalized 3D tumor architecture, which
further analyses are needed to confirm this statement. is widely recognized to significantly influence behavior of
Human pancreatic ductal epithelial cells were seeded cancer cells. 22,85,86 Moreover, the crosstalk between stromal
in the cavity of the structures, where HFF1 were allowed and epithelial cells can also be easily monitored in this
to grow for 2 weeks (Figure 5). The epithelial–stromal cells model, as the open structure allows the observation of
crosstalk occurring in the MEW model was studied in terms the epithelial cell organization within the cavity, between
of fibroblasts inflammation mediated by IL-6 (Figure 5b). the fibroblast interconnections (Figure 7). Although these
Indeed, the release of IL-6 by inflamed tumor-associated results demonstrate the possibility to model the functional
fibroblasts plays a key role in PDAC–stroma interplay unit of the exocrine pancreas in vitro and to study the
and regulates a wide range of mechanisms involved in interactions between PDAC and stromal cells occurring
pancreatic cancer, such as angiogenesis, epithelial-to- at different stages of pancreatic cancer progression in a
mesenchymal transition, and immunosuppression. 71-73 It very controlled and biomimetic way, additional works are
has been demonstrated previously that the release of IL-6 warranted to improve this model. For instance, advanced
by HPDE-KRAS cells under monoculture on artificial bioprinting systems can be employed in combination with
87
substrates was minimal and negligible compared to HFF1. MEW to print the epithelial cells into a monolayer on the
21
Our results indicate a higher IL-6 release by fibroblasts in previously obtained MEW model, within the acinar-like
co-culture with HPDE-KRAS cells for 2 days and 3 days, cavity in a precise and software-guided way.
in comparison with HFF1 alone or HFF1 under co-culture
with healthy HPDE cells (HPDE-WT). This is in line with 5. Conclusion
studies in literature reporting the role of the KRAS oncogene This work describes the engineering approach adopted
as a driver for the IL-6 production by stromal cells. 74,75 to fabricate a layer-by-layer microscale model resembling
The MEW model developed in this study recapitulates the half structure of the functional unit of the exocrine
the in vivo pathological condition, accompanied by pancreas, for the study of pancreatic cancer. MEW was
IL-6 secretion by fibroblasts during the inflammation of employed to obtain the complex 3D structure without the

Volume 10 Issue 2 (2024) 425 doi: 10.36922/ijb.1975