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International Journal of Bioprinting 3D-bioprinted respiratory disease model

limiting the mobility and communication of incorporated reported to provide good physiological relevance, thus
cells. The addition of low concentrations of components increasing the applicability of these engineered models.
that may aid in cellular remodeling and dissolution Similar trends can be observed when comparing the results
of the matrix material, such as sodium citrate, may be obtained in this study to previous literature on respiratory
considered; however, this would have to be balanced with disease models; though it should be noted that different
maintaining the structural integrity of the constructs to cell sources and biomaterials have been used across
limit degradation for long-term use. studies, resulting in some variance. The viral titer obtained

The 3D-bioprinted constructs are designed in a way to can be compared with viral mRNA and viral titers tracked
create an epithelial surface layer within a contained pool. in other respiratory model studies with the same trend, i.e.,
Infection begins with the HBEpC barrier layer, followed by a significant increase at 24 HPI before remaining relatively
39,41
the virus progressing into the bulk of the material. Infection steady to slightly decreasing by 48 h. The XTT results
of the 3D constructs caused a slight decrease in cellular were also quite similar, with cellular metabolism slightly
metabolism, with clusters of dead cells visible in live/ decreasing over the infection period. The upregulation of
dead imaging, demonstrating a clustered infection pattern mRNAs related to IL-29, IL-8, and IL-1β is also similar to
similar to that observed in clinical infections and previous the literature, though a direct comparison cannot be made
42
studies. The 2D and 3D PCR results displayed consistent due to different methods of measurement.
41
trends, with the 3D infection results demonstrating lower Overall, the cumulation of results indicates that the
mRNA fold changes overall. However, IL-29 mRNA was alginate/gelatin/collagen biomaterial is a favorable material
upregulated to the greatest extent in both conditions, for bioprinting and long-term culture of respiratory tissue
with IP-10 mRNA also significantly upregulated in all constructs. The inclusion of a biomechanical stimulus in
conditions. One notable difference between the 2D and the form of a bioreactor mimicking the airflow and pressure
3D experiments is the extent of IL-8 mRNA upregulation, conditions of breathing increases cellular metabolism.
as the 2D studies demonstrated a ~550-fold increase in This can be further increased through the inclusion of
IL-8 mRNA that was not evident at any stage of the 3D hepatocyte growth factor-loaded nanoparticles to generate
infection study, though slight upregulation of IL-8 mRNA a highly viable cellular construct. Infection of these
in the dynamic and static condition was recorded. When 3D-bioprinted constructs designed to have a contained,
comparing differences in cytokine release between static, air-exposed epithelial barrier layer can easily be carried
dynamic, and dynamic with nanoparticle conditions, out through the addition of the infectious agent to the
the differences are greatest at the 6-h timepoint, with the pool to mimic biological infection pathways; the results
nanoparticle-containing constructs upregulating IL-29 obtained were similar to that of the 2D experiments. It
mRNA and IP-10 mRNA to a greater extent early in the is indicated that the inclusion of biomechanical stimulus
infection period. As nanoparticles are incorporated before when culturing infected constructs affects cytokine gene
printing and infection experiments are not conducted until expression, while an increase in epithelial cell numbers
14 days of culture (7 days pre-seeding; 7 days post-seeding), caused by nanoparticle incorporation in the biomaterial
it is likely that the influence of incorporating nanoparticles may lead to increased cytokine gene expression at the early
is limited to increasing epithelial cell proliferation during stages of infection.
culture, leading to an increased number of epithelial cells. The scope of this research was to determine whether
This would cause a greater upregulation in cytokine release the inclusion of a biomechanical stimulus and a controlled
early in the infection before the virus has penetrated the release system for growth factors is worth pursuing for
underlying fibroblast cells in the construct. By the 24 HPI, the further development of respiratory tissue models
most differences fall within standard deviation; however, in infection studies. While this study successfully
there is a general trend of the static condition, resulting demonstrates the importance of both factors, this scope did
in greater up-regulation of cytokine mRNAs, such as IL- result in some limitations. For example, the microbiological
29 and IP-10, in comparison to both dynamic conditions. characterization of cell behavior within this study was
This demonstrates that the inclusion of a biomechanical limited, and the use of undifferentiated THP-1 cells only
stimulus may influence viral infection.
functions as a monocytic immune component, which lacks
The three-layer design of the present model shares relevance to alveolar and other relevant macrophages.
similarities to other models reported in the literature, as Further work related to this should focus on enhancing
many aim to form a surface epithelial barrier layer over- the relevance of the immune component and conducting
laying fibroblasts, with alveolar models reported in the further characterizations on the differences in cell behavior,
literature often also incorporating endothelial cells as the including flow cytometry and cell transcriptomes, in both
bottom layer. 41,42,52 These multi-layer designs have been dynamic and static conditions.

Volume 10 Issue 6 (2024) 426 doi: 10.36922/ijb.3895