Page 449 - IJB-9-5

International Journal of Bioprinting Core-shell bioarchitectures

lower than the α = 0.6 threshold, while it increased with other pathophysiological conditions, such as lung fibrosis
increasing Pluronic concentration. Experimental tests and aging, by appropriately tuning material parameters
confirmed that the presence of Pluronic was essential in (e.g., viscosity, stiffness) [38-41] . The strategy could also be
the formation of the liquid-core structures. Pluronic is a exploited to generate stratified structures, such as tumors
copolymer of hydrophilic and hydrophobic groups which, and embryonic layers [42,43] , or more advanced systems
at the concentrations employed here, forms micellar mimicking the curved topology of interfaces between
structures in aqueous solutions; hence, the entanglement tissues occurring in different organs in the human
between alginate or Pluronic chains can be neglected . body [42,44,45] . Finally, more in-depth cell studies to better
[37]
The steric hindrance and increased core viscosity offered understand cell response to curvature could be conducted
by the micelles likely hamper alginate diffusion into the by tuning the extrusion parameters highlighted in this
liquid core. work to modulate core–shell radii [41,42] .

The experimental results also highlighted that the
relative core–shell extrusion flow rate is fundamental for 5. Conclusion
bioprinting core–shell structures with optimal roundness. An innovative in silico–in vitro strategy was developed
For solid and liquid cores, the shell extrusion rate should for bioprinting-structured spheroids able to replicate the
be higher than that of the core, promoting the formation multilayer tissue organization and the interface between
of a uniform shell layer. Although challenging, with some epithelial (or endothelial) tissues and fluid (either liquid
of these flow rate combinations, we also achieved the or air) lumens. The computational tools enable the
formation of an air-core. To the best of our knowledge, definition of a working window, optimizing material
the spontaneous formation of an air-containing lumen has combination and extrusion parameters. The generation of
never been observed in bioprinted structures. This result is core–shell spheroids with our bioprinting set-up presents
thus particularly relevant for lung models. a significant advantage with respect to standard culture
Our preliminary cellular studies demonstrated the methods allowing the rapid fabrication of complex and
cytocompatibility of the bioprinting process and the physiologically relevant architectures. Our results show
feasibility of encapsulating different cell types in a core– improved barrier properties with respect to controls,
shell construct. The alveolar microenvironment was contributing to the establishment of human-relevant
effectively recapitulated, with epithelial cells lining the in vitro models, in line with 3Rs principles (refinement,
lumen and fibroblasts in the shell, representing interstitial reduction, and replacement of animal experiments).
spaces in the lung extracellular matrix. In terms of
architecture, the epithelial cells are exposed to a radius Acknowledgments
of curvature = 1.01 ± 0.12 mm, while fibroblasts perceive None.
a radius of curvature which varies with their position in
the shell, ranging from 1.5 ± 0.11 mm to 1.01 ± 0.12 mm. Funding
With an elastic modulus in the kPa range (see Section S9
in Supplementary File), these constructs are also able This research received funding from the Italian Ministry
to mimic healthy matrix conditions, thus maintaining of Health for the definition alternatives to animal-based
fibroblasts in a quiescent state without the onset of fibrotic research.
processes . The reduction of FITC-dextran passage with
[38]
respect to the cell-free blanks suggests that the presence of Conflict of interest
cells hindered passive diffusion, which may be linked to The authors declare no conflicts of interest.
the onset of barrier formation. The increase of rhodamine
passage with respect to the blanks is particularly Author contributions
interesting since it indicates active transcellular transport
and epithelial cell polarization. Further studies may Conceptualization: Ludovica Cacopardo, Arti Ahluwalia
improve uniformity of the epithelial layer along all the Investigation: Nicole Guazzelli, Ludovica Cacopardo
lumen (e.g., by improving the composition and porosity Methodology: Nicole Guazzelli, Ludovica Cacopardo,
of the shell matrix and by implementing an air–liquid Alessandro Corti, Arti Ahluwalia
interface thanks to air-filled cores fabrication). The Formal analysis: Nicole Guazzelli, Ludovica Cacopardo
presence of fibroblasts does not significantly affect the Writing – original draft: Nicole Guazzelli, Ludovica
parameters investigated. However, since fibroblasts are Cacopardo, Arti Ahluwalia
fundamental for lung tissue homeostasis, in future studies, Writing – review: Nicole Guazzelli, Ludovica Cacopardo,
the co-culture model could be used for recapitulating Alessandro Corti, Arti Ahluwalia

Volume 9 Issue 5 (2023) 441 https://doi.org/10.18063/ijb.771